Applied Biosystems StepOnePlus, StepONE User Manual

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Page 1

Reagent Guide

Applied Biosystems StepOne™ and StepOnePlus

™

Real-Time PCR Systems

Page 2

Page 3

Applied Biosystems StepOne™ and

™

StepOnePlus

Real-Time PCR

Systems

Reagent Guide

Page 4

Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document.

APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPE CIAL, INCIDENTAL, IN DIRECT, PU NI TIVE, MULTIPLE OR CONSEQU ENTIAL DAMAG ES IN CONNEC TI ON WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

For R ese arc h U se On ly. Not for us e in dia gn ost ic p roc edu res .

NOTICE TO PURCHASER: Label License

The StepOne™ and StepOnePlus™ Real-Time PCR Systems are covered by US patents and corresponding claims in their non-US counterparts, owned by Applied Biosystems Inc. No right is conveyed expressly, by implication, or by esto ppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5′ nuclease methods. Further infor mation on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

NOTICE TO PURCHASER:

PLEASE REFER TO THE USER'S GUIDE OR PRODUCT INSERT OF THE REAGENTS NAMED HEREIN FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION.

TRADEMARKS:

Applied Biosystems, AB (Design), MicroAmp, Primer Express, and VIC are registered trademarks, and FAM, JOE, MicroAmp, MultiScribe, NED, ROX, StepOne, StepOnePlus, TAMRA, TET, and VeriFlex are trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries.

AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.

SYBR is a registered trademark of Molecular Probes, Inc.

All other trademarks are the sole property of their respective owners.

Part Number 4379704 Rev. E

06/2010

Page 5

Contents Front Cover

Preface

How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Chapter 1 Introduction

About the StepOne™ and StepOnePlus™ Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Supported Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Preparing for Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6

Select the Experiment Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7

Select the Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8

Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9

Chapter 2 Reagent Overview

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Ta qM an

SYBR

Selecting the Appropriate Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Minimizing DNA Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Green Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

Chapter 3 Quantitation Experiments

Section 3.1: About Quantitation Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Select a Quantitation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5

Select 1-Step or 2-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8

Select Singleplex or Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10

Select the Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-12

Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-12

Section 3.2: Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16

TaqMan

Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16

iiiApplied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

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Custom TaqMan® Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18

Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19

Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21

Design Primers and Probes Using Primer Express

Select the Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24

Use the Recommended Thermal Cycling Conditions . . . . . . . . . . . . . . . . . . . . . . 3-27

Optimize Primer Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-30

Optimize the Probe Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-33

For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-35

Chapter 4 Genotyping Experiments

Section 4.1: About Genotyping Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Section 4.2: Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Pre-Designed/Validated Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

TaqMan

Pre-Developed TaqMan

Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Custom TaqMan

Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16

Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16

SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

Drug Metabolism Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11

Assay Reagents for Allelic Discrimination . . . . . . . . . . 4-12

SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Software . . . . . . . . . . . . . . . 3-21

Chapter 5 Presence/Absence Experiments

Section 5.1: About Presence/Absence Experiments . . . . . . . . . . . . . . . . . . . . . . . . . 5-3

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4

Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6

Section 5.2: Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10

TaqMan

Custom TaqMan

TaqMan

Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15

Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16

iv Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10

Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12

Exogenous Internal Positive Control Reagents . . . . . . . . . . . . . . . . . . . 5-13

Page 7

Appendix A Formulas

Standard Deviation Calculation Using the Standard Curve Method . . . . . . . . . . . . . . . A-2

Standard Deviation Calculation Using the Comparative Method . . . . . . . . . . . . . . . . . A-5

Formula for Comparative C

(ΔΔCT) Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7

Appendix B Primer Limiting in Multiplex PCR

Appendix C Assay Design Guidelines

Appendix D Reagent Part Numbers

Quantitation Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

Genotyping Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5

Pre-Designed/Validated Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-5

Presence/Absence Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-7

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-7

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-8

Bibliography

Glossary

Index

vApplied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

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vi Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

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Preface

How to Use This Guide

About the System

Documentation

The guides listed below are shipped with the Applied Biosystems StepOne™ and StepOnePlus

Guide Purpose and Audience PN

Applied Biosystems StepOne StepOnePlus

™

Real-Time PCR Systems

™

and

Getting Started Guide for Genotyping Experiments

™

Applied Biosystems StepOne StepOnePlus

™

Real-Time PCR Systems

and

Getting Started Guide for Presence/Absence Experiments

™

Applied Biosystems StepOne StepOnePlus

™

Real-Time PCR Systems

and

Getting Started Guide for Relative

™

and

Standard Curve and Comparative C Experiments

Applied Biosystems StepOne StepOnePlus

™

Real-Time PCR Systems Getting Started Guide for Standard Curve Experiments

™

Applied Biosystems StepOne

and StepOnePlus™ Real-Time PCR Systems Installation, Networking, and Maintenance Guide

™

Applied Biosystems StepOne

and StepOnePlus™ Real-Time PCR Systems Installation Quick Reference Card

Applied Biosystems StepOne

™

and StepOnePlus™ Real-Time PCR Systems Reagent Guide

™

Real-Time PCR Systems (StepOne™ and StepOnePlus™ systems).

Explains how to perform experiments on the StepOne and

4376786 StepOnePlus systems. Each Getting Started Guide functions as both:

• A tutorial, using example experiment data provided with

the Applied Biosystems StepOne Software (StepOne™ software).

™

Real-Time PCR

4376787

• A guide for your own experiments.

Intended for laboratory staff and principal investigators who perform experiments using the StepOne or StepOnePlus system.

4376785

4376784

Explains how to install and maintain the StepOne and

4376782 StepOnePlus systems.

Intended for laboratory staff responsible for the installation and maintenance of the StepOne or StepOnePlus system

4376783

Provides information about the reagents you can use on the

4379704 StepOne and StepOnePlus systems, including:

• An introduction to TaqMan

and SYBR® Green reagents

• Descriptions and design guidelines for the following

experiment types: – Quantitation experiments – Genotyping experiments – Presence/absence experiments

Intended for laboratory staff and principal investigators who perform experiments using the StepOne or StepOnePlus system.

viiApplied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

Page 10

Preface

Guide Purpose and Audience PN

Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Site Preparation Guide

™

Applied Biosystems StepOne PCR Software Help

Assumptions

Real-Time

This guide assumes that you:

• Are familiar with the Microsoft Windows

• Are familiar with the Internet and Internet browsers.

• Know how to handle DNA and/or RNA samples and prepare them for PCR.

• Understand data storage, file transfer, and copying and pasting.

• Have networking experience, if you plan to integrate the StepOne or StepOnePlus system into your existing laboratory data flow.

Explains how to prepare your site to receive and install the StepOne and StepOnePlus systems.

Intended for personnel who schedule, manage, and perform the tasks required to prepare your site for installation of the StepOne or StepOnePlus system.

Explains how to use the StepOne software to:

• Set up, run, and analyze experiments using the StepOne and StepOnePlus systems.

• Monitor networked StepOne and StepOnePlus instruments.

• Calibrate StepOne and StepOnePlus instruments.

• Verify the performance of StepOne and StepOnePlus instruments with an RNase P run.

Intended for:

• Laboratory staff and principal investigators who perform experiments using the StepOne or StepOnePlus system.

• Laboratory staff responsible for the installation and maintenance of the StepOne or StepOnePlus system.

XP operating system.

4376768

Te xt Co n ve n ti o ns

This guide uses the following conventions:

• Bold text indicates user action. For example: Ty p e 0, then press Enter for each of the remaining fields.

• Italic text indicates new or important words and is also used for emphasis. For example:

Before analyzing, always prepare fresh matrix.

• A right arrow symbol () separates successive commands you select from a drop-down or shortcut menu. For example:

Select FileOpen.

User Attention

Words

Two user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below:

Note: – Provides information that may be of interest or help but is not critical to the

use of the product.

IMPORTANT! – Provides information that is necessary for proper instrument

operation, accurate reagent kit use, or safe use of a chemical.

viii Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

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Examples of the user attention words appear below:

Note: The Calibrate function is also available in the Control Console.

IMPORTANT! To verify your client connection, you need a valid user ID.

How to Obtain More Information

Preface

Documentation

Other StepOne and StepOnePlus System Documents

The documents listed in the table below are not shipped with the StepOne or StepOnePlus instrument.

Document PN

™

Applied Biosystems StepOne

and StepOnePlus™ Real-Time PCR

Systems Installation Performance Verification Protocol

™

Applied Biosystems StepOne

and StepOnePlus™ Real-Time PCR

Systems Installation Qualification-Operation Qualification Protocol

Applied Biosystems StepOne

™

and StepOnePlus™ Real-Time PCR

Systems Planned Maintenance Protocol

Documents Related to Genotyping Experiments

Document PN

Allelic Discrimination Pre-Developed TaqMan Reference Card

Custom TaqMan

Ordering TaqMan

Performing a Custom TaqMan

Genomic Assays Protocol 4367671

SNP Genotyping Assays Protocol 4334431

SNP Genotyping Assays Quick Reference Card 4374204

SNP Genotyping Assay for 96-Well

Plates Quick Reference Card

Performing a TaqMan

Drug Metabolism Genotyping Assay for 96-Well

Plates Quick Reference Card

Pre-Developed TaqMan

Assay Reagents Allelic Discrimination

Protocol

TaqM an

Drug Metabolism Genotyping Assays Protocol 4362038

SNP Genotyping Assays Protocol 4332856

Assay Reagents Quick

4376791

4376790

4376788

4312212

4371394

4367636

4312214

ixApplied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

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Preface

Documents Related to Presence/Absence Experiments

Document PN

DNA Isolation from Fresh and Frozen Blood, Tissue Culture Cells, and

4343586

Buccal Swabs Protocol

NucPrep® Chemistry: Isolation of Genomic DNA from Animal and Plant

4333959

Tissue Protocol

PrepMan

Documents Related to Relative Standard Curve and Comparative C

Ultra Sample Preparation Reagent Protocol 4318925

Experiments

Document PN

Amplification Efficiency of TaqMan

Gene Expression Assays

127AP05

Application Note

Applied Biosystems High-Capacity cDNA Reverse Transcription Kits

4375575

Protocol

Custom TaqMan

Primer Express

TaqM an

Gene Expression Assays Protocol 4333458

Gene Expression Assays Protocol 4334429

Software Version 3.0 Getting Started Guide 4362460

User Bulletin #2: Relative Quantitation of Gene Expression 4303859

Documents Related to Standard Curve Experiments

Document PN

Amplification Efficiency of TaqMan

Gene Expression Assays

Application Note

Custom TaqMan

Primer Express

TaqM an

Gene Expression Assays Protocol 4334429

Software Version 3.0 Getting Started Guide 4362460

Gene Expression Assays Protocol 4333458

User Bulletin #2: Relative Quantitation of Gene Expression 4303859

127AP05

x Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

Page 13

Documents Related to the Reagent Guide

Document PN

Preface

Applied Biosystems High-Capacity cDNA Reverse Transcription Kits Protocol

Custom TaqMan

Power SYBR

Pre-Developed TaqMan

Gene Expression Assays Protocol 4334429

Genomic Assays Protocol: Submission Guidelines 4367671

SNP Genotyping Assays Protocol 4334431

Green PCR Master Mix and RT-PCR Protocol 4367218

Assay Reagents Allelic Discrimination

Protocol

Primer Express

Green PCR and RT-PCR Reagents Protocol 4304965

SYBR

Green PCR Master Mix and RT-PCR Reagents Protocol 4310251

TaqM an

Software Version 3.0 Getting Started Guide 4362460

Drug Metabolism Genotyping Assays Protocol 4362038

Exogenous Internal Positive Control Reagents Protocol 4308335

Fast Universal PCR Master Mix (2✕) Protocol 4351891

Gene Expression Assays Protocol 4333458

Gene Expression Master Mix Protocol 4371135

Genotyping Master Mix Protocol 4371131

SNP Genotyping Assays Protocol 4332856

Universal PCR Master Mix Protocol 4304449

4375575

4312214

Obtaining

Information from

the Software Help

User Bulletin #2: Relative Quantitation of Gene Expression 4303859

Using TaqMan

Endogenous Control Assays to Select an Endogenous

127AP08

Control for Experimental Studies Application Note

For more documentation, see “How to Obtain Support” on page xii.

Note:

The StepOne Software Help describes how to use each feature of the user interface. Access the Help from within the software by doing one of the following:

•Press F1.

• Click in the toolbar.

• Select HelpStepOne Software Help.

To find topics of interest in the Help:

• Review the table of contents.

• Search for a specific topic.

• Search an alphabetized index.

xiApplied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

Page 14

Preface

Send Us Your

Comments

Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can e-mail your comments to:

[email protected]

IMPORTANT! The e-mail address above is only for submitting comments and

suggestions relating to documentation. To order documents, download PDF files, or for help with a technical question, go to http://www.appliedbiosystems.com, then click the link for Support. (See “How to Obtain Support” on page Front Matter-xii).

How to Obtain Support

For the latest services and support information for all locations, go to

http://www.appliedbiosystems.com, then click the link for Support.

At the Support page, you can:

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support

• Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents

• Download PDF documents

• Obtain information about customer training

• Download software updates and patches

In addition, the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities.

IMPORTANT! When directed to do so by this guide, or when you need to schedule

maintenance for your StepOne planned maintenance or temperature verification/calibration), contact the Applied Biosystems Care Center. To obtain a phone number for or to send an email to the center, go to http://www.appliedbiosystems.com/support/contact.

™

or StepOnePlus™ instrument (such as annual

xii Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

Page 15

Introduction 1

This chapter covers:

About the StepOne

Supported Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Preparing for Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6

Select the Experiment Type. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7

Select the Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8

Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9

™

and StepOnePlus™ Systems . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

1-1Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

Page 16

Chapter 1 Introduction

About the StepOne™ and StepOnePlus™ Systems

There are two models available for this Real-Time PCR System:

System Features

About Data

Collection

Applied Biosystems StepOne PCR System (StepOne™ system)

Applied Biosystems StepOnePlus™ RealTime PCR System (StepOnePlus

™

Real-Time

™

system)

• 48-well platform

• Three-color system

• 96-well platform

• Four-color system

•VeriFlex

™

sample blocks

The StepOne and StepOnePlus systems use fluorescent-based polymerase chain reaction (PCR) reagents to provide:

• Quantitative detection of target nucleic acid sequences (targets) using real-time analysis.

• Qualitative detection of targets using post-PCR (endpoint) analysis.

• Qualitative analysis of the PCR product (achieved by melt curve analysis that occurs post-PCR).

The StepOne and StepOnePlus systems collect raw fluorescence data at different points during a PCR, depending on the type of run that the instruments perform:

Run Type Data Collection Point

Real-time runs Standard curve The instrument collects data following each

Relative standard curve

extension step of the PCR.

Comparative C

)

(ΔΔC

Post-PCR (endpoint) runs

Genotyping The instrument collects data:

Presence/absence

Regardless of the run type, a data collection point or read on the StepOne StepOnePlus

™

instrument consists of three phases:

• Before the PCR (For presence/absence experiments, data collection before the PCR is optional, but recommended.)

• (Optional) During the PCR. The instrument can collect data during the run (real-time); collecting data during the run can be helpful for troubleshooting.

•After the PCR

™

1. Excitation – The instrument illuminates all wells of the reaction plate within the instrument, exciting the fluorophores in each reaction.

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Page 17

Chapter 1 Introduction

2. Emission – The instrument optics collect the residual fluorescence emitted from the wells of the reaction plate. The resulting image collected by the device consists only of light that corresponds to the range of emission wavelengths.

About the Filters

3. Collection – The instrument assembles a digital representation of the residual fluorescence collected over a fixed time interval. The StepOne

™

software stores

the raw fluorescent image for analysis.

After a run, the StepOne software uses calibration data (spatial, dye, and background) to determine the location and intensity of the fluorescent signals in each read, the dye associated with each fluorescent signal, and the significance of the signal.

The StepOne and StepOnePlus systems use the following filters:

StepOne system StepOnePlus system

Filter Dye Filter Dye

1FAM

2JOE™ dye 2 JOE™ dye

3ROX

™

dye 1 FAM™ dye

Green dye SYBR® Green dye

SYBR

dye VIC® dye

VIC

™

dye 3 TAMRA™ dye

NED™ dye

4ROX

™

dye

About the

VeriFlex

™

Te ch no lo gy

For More

Information

The StepOnePlus instrument contains six independently thermally regulated Ve ri Fl ex

™

blocks to help you optimize your thermal cycling conditions. You can set a different temperature for one or more of the VeriFlex blocks, creating up to six different zones for samples, or you can set the same temperature for each of the VeriFlex blocks.

For more information about any of the topics discussed in this guide, access the Help from within Applied Biosystems StepOne

™

Real-Time PCR Software v2.0 by

pressing F1, clicking in the toolbar, or selecting HelpStepOne Software Help.

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Chapter 1 Introduction

# Consumable

AMicroAmp™ Fast Optical 48-Well Reaction Plate

BMicroAmp

™

Fast 48-Well Tray

CMicroAmp™ 96-Well Support Base

DMicroAmp

™

Optical 8-Cap Strip

EMicroAmp

™

Fast 8-Tube Strip

FMicroAmp® Fast Reaction Tube with Cap

GMicroAmp

™

48-Well Optical Adhesive Film

HMicroAmp

™

48-Well Base Adaptor

Supported Consumables

StepOne System

The StepOne system supports the consumables listed below. These consumables are for use with both standard and Fast reagents/protocols.

IMPORTANT! Use only Fast consumables (reaction plates, tube strips, and tubes)

with the StepOne and StepOnePlus systems, even when performing an experiment with standard reagents.

Consumable Part Number

•MicroAmp

•MicroAmp™ 96-Well Support Base

™

Fast Optical 48-Well Reaction Plate

™

48-Well Optical Adhesive Film

• 4375816

• 4375323 and 4375928

™

Fast 8-Tube Strip

™

Optical 8-Cap Strip

Fast Reaction Tube with Cap • 4358297

™

Fast 48-Well Tray

™

48-Well Base Adaptor

• 4358293

• 4323032

• 4375282

• 4375284

• 4379590

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Chapter 1 Introduction

# Consumable

AMicroAmp™ Fast Optical 96-Well Reaction Plate

BMicroAmp™ 96-Well Tray for VeriFlex™ Blocks

CMicroAmp

™

96-Well Support Base

DMicroAmp

™

Optical 8-Cap Strip

EMicroAmp™ Fast 8-Tube Strip

FMicroAmp

Fast Reaction Tube with Cap

GMicroAmp

™

Optical Adhesive Film

StepOnePlus

System

The StepOnePlus system supports the consumables listed below. These consumables are for use with both standard and Fast reagents/protocols.

IMPORTANT! Use only Fast consumables (reaction plates, tube strips, and tubes)

with the StepOne and StepOnePlus systems, even when performing an experiment with standard reagents.

Consumable Part Number

•MicroAmp™ Fast Optical 96-Well Reaction Plate with Barcode

•MicroAmp™ Optical Adhesive Film

™

•MicroAmp

Fast 8-Tube Strip

•MicroAmp™ Optical 8-Cap Strip

•MicroAmp

Fast Reaction Tube with Cap • 4358297

™

96-Well Tray for VeriFlex™ Blocks

•MicroAmp™ 96-Well Support Base

™

•MicroAmp

Adhesive Film Applicator

•MicroAmp™ Cap Installing Tool (Handle)

• 4346906 and 4366932

• 4360954 and 4311971

• 4358293

• 4323032

• 4379983

• 4379590

• 4333183

• 4330015

1-5Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

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Chapter 1 Introduction

Preparing for Experiments

General Workflow

Information in

This Guide

Before performing experiments on the StepOne and StepOnePlus systems, prepare for the experiment as follows:

1. Select an experiment type (page 1-7).

2. Select the reagent type (page 1-8).

3. Select the assay type (page 1-9).

This chapter provides general information on the experiment types, reagent types, and assay types you can use with the StepOne and StepOnePlus systems.

Subsequent chapters provide specific information:

Chapter Description

Chapter 2, Reagent Overview • Describes and compares the TaqMan® and

• Provides information on minimizing DNA

Chapter 3, Quantitation Experiments

Chapter 4, Genotyping Experiments

• Explains how the experiment type works.

• Provides a specific workflow for the experiment

• Provides design guidelines for each assay type.

SYBR

contamination.

type.

Green reagents.

Chapter 5, Presence/Absence Experiments

1-6 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

Page 21

Select the Experiment Type

In this guide, the term experiment refers to the entire process of performing a run using the StepOne or StepOnePlus system, including setup, run and analysis. You can perform the following types of experiments on the StepOne and StepOnePlus systems:

• Quantitation, including: – Standard curve – Relative standard curve – Comparative C

•Genotyping

• Presence/absence

Note: You can also perform melt curve analysis on the StepOne and StepOnePlus

systems. For more information, access the Help from within the StepOne software by clicking in the toolbar or pressing F1.

(ΔΔCT)

Chapter 1 Introduction

Endpoint vs.

Real-Time

Experiments

The three experiment types can be categorized as real-time or endpoint experiments, as described below.

Category Properties Experiment Type

Real-time • The instrument monitors the progress of the

PCR as it occurs.

• Data are collected throughout the PCR process.

• Reactions are characterized by the point in time during cycling when amplification of a target is first detected.

Endpoint • Data are collected at the end of the PCR

process.

• Reactions are characterized by the quantity of the target accumulated at the end of PCR.

• The datapoint is the normalized intensity of the reporter dye, or R

‡

Note: Some endpoint experiments also include

pre-PCR datapoints. If so, the system calculates the delta R formula:

ΔRn = Rn (post-PCR read) – Rn (pre-PCR read)

where Rn = normalized reporter

(ΔRn) value per the following

Quantitation

Genotyping

Presence/absence

‡ Kwok and Higuchi, 1989.

§Saiki et al., 1985.

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Chapter 1 Introduction

Select the Reagent Type

You can use the following reagent types (chemistries) on the StepOne and StepOnePlus systems:

•TaqMan

• SYBR

• Other fluorescent-based reagents

reagents

Green reagents

Taq Ma n

Reagents

SYBR Green

Reagents

TaqMan reagents include Applied Biosystems TaqMan® assays (preformulated mixes that contain probe and primer sets) and Applied Biosystems TaqMan

master

mixes. You can use TaqMan reagents for the following experiment types:

• Quantitation, including: – Standard curve – Relative standard curve – Comparative C

(ΔΔCT)

•Genotyping

• Presence/absence

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

SYBR Green reagents include primers and master mixes that contain SYBR® Green dye. You can use SYBR Green reagents for quantitation experiments:

• Standard curve

• Relative standard curve

• Comparative C

Note: You cannot perform multiplex PCR using SYBR Green reagents. For more

(ΔΔCT)

information, see “Select Singleplex or Multiplex PCR” on page 3-10.

Other Reagents

You can use other fluorescent-based reagents on the StepOne and StepOnePlus systems, but note the following when using the StepOne software:

• You must design your experiment using Advanced Setup instead of the Design Wizard.

• For Applied Biosystems TaqMan and SYBR Green reagents, the StepOne software automatically calculates reaction volumes in the Reaction Setup screen.

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Page 23

Select the Assay Type

In the StepOne software, you can select the following assay types for quantitation, presence/absence, and genotyping experiments:

Exp e r i me n t Ty pe Ass a y Ty pe See . . .

Chapter 1 Introduction

Quantitation and

Presence/

Absence

Experiments

Quantitation

Presence/absence

Genotyping • Pre-Designed/Validated

‡ Quantitation experiments include standard curve, relative standard curve, and

comparative C

Inventoried/Made to Order Assays

‡

experiments.

• Inventoried/Made to Order

•Custom

•User-Designed

below

page 1-10

For quantitation or presence/absence experiments, select the Inventoried/Made to Order assay type in the StepOne software if you are using:

• Taq Ma n labeled TaqMan

Gene Expression Assays, Inventoried – Predesigned FAM™ dye-

MGB (minor groove binder) probe and primer sets that can be purchased off-the-shelf. The assay mix is available in a single, preformulated 20✕ tube.

• Taq Ma n

Gene Expression Assays, Made to Order – Predesigned FAM dye-

labeled TaqMan MGB probe and primer sets that are manufactured at the time of order. The assay mix is available in a single, preformulated 20✕ tube.

• Custom TaqMan

Gene Expression Assays – FAM dye-labeled TaqMan

MGB probe and primer sets that are designed, synthesized, and formulated by the Custom TaqMan

Genomic Assays service based on sequence information that you submit. The assay mix is available in a single, preformulated 20✕ or 60✕ tube.

Note: To select the assay type in the StepOne software, go to the Reaction Setup

screen in either the Design Wizard or Advanced Setup, then select Inventoried/Made to Order from the Assay Type dropdown menu.

Custom Assays

For quantitation and presence/absence experiments, select the Custom assay type in the StepOne software if you are designing your own assays (primers and probes) with Primer Express

Software and TaqMan or SYBR Green reagents. When designing your own assays, follow the Applied Biosystems Assay Design Guidelines to optimize results.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

Note: To select the assay type in the StepOne software, go to the Reaction Setup

screen in either the Design Wizard or Advanced Setup, then select Custom from the Assay Type dropdown menu.

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Chapter 1 Introduction

Genotyping

Experiments

Pre-Designed/Validated Assays

For genotyping experiments, the Pre-Designed/Validated assay type includes:

• Taq Ma n labeled TaqMan available as TaqMan

SNP Genotyping Assays – Predesigned FAM™ dye- and VIC® dye-

MGB (minor groove binder) probes and primer sets that are

Pre-Designed SNP Genotyping Assays. The TaqMan PreDesigned SNP Genotyping Assays are manufactured at the time of order (Made to Order). The assay mix is available in a single, preformulated 20✕ tube.

• Taq Ma n

Drug Metabolism Genotyping Assays – Predesigned FAM dye- and

VIC dye-labeled TaqMan MGB probes and primer sets that can be purchased off-the-shelf (Inventoried). The assay mix is available in a single, preformulated 20✕ tube.

• Pre-Developed TaqMan

(TaqMan

PDARs for AD) – Predesigned FAM dye- and VIC dye-labeled

Assay Reagents for Allelic Discrimination

TaqMan MGB probes and primer sets that can be purchased off-the-shelf (Inventoried). The assay mix is available in a single, preformulated 10✕ tube.

Note: To select a SNP assay in the StepOne software, go to the SNP Assays screen

in the Design Wizard or to the Plate Setup screen in Advanced Setup. In the SNP Assays screen or Plate Setup screen, select an assay from the library or create a new assay.

Custom Assays

For genotyping experiments, the Custom assay type includes the Custom TaqMan® SNP Genotyping Assays. The Custom TaqMan SNP Genotyping Assays are FAM dye- and VIC dye-labeled TaqMan MGB probes and primer sets that are designed, synthesized, and formulated by the Custom TaqMan

Genomic Assays service based on sequence information that you submit. The assay mix is available in a single, preformulated 40✕ or 80✕ tube.

Note: To select a SNP assay in the StepOne software, go to the SNP Assays screen

in the Design Wizard or to the Plate Setup screen in Advanced Setup. In the SNP Assays screen or Plate Setup screen, you can select an assay from the library or create a new assay.

User-Designed Assays

If you want to design your own primers and probes for SNP assays, refer to the

Primer Express

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

Software Version 3.0 Getting Started Guide.

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

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Page 25

Reagent Overview 2

This chapter covers:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Taq Ma n

SYBR

Selecting the Appropriate Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Minimizing DNA Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Green Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

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Page 26

Chapter 2 Reagent Overview

Overview

Applied Biosystems has developed two reagent types (chemistries) that can be used to detect PCR products on the Applied Biosystems StepOne Real-Time PCR Systems:

•TaqMan

• SYBR

TaqMan® Reagents

Experiment

Ty pe s

Taq Ma n® reagents include Applied Biosystems TaqMan® assays (preformulated mixes that contain probe and primer sets) and Applied Biosystems TaqMan mixes. The assays are specific to the target of interest. The master mixes contain the remaining components needed for the PCR reaction. You can use TaqMan reagents for the following experiment types:

• Quantitation, including: – Standard curve – Relative standard curve – Comparative C

•Genotyping

• Presence/absence

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

reagents (below)

Green reagents (page 2-4)

(ΔΔCT)

™

and StepOnePlus™

™

master

dye

Development of

Taq Ma n

Reagents

Initially, intercalator dyes were used to measure real-time PCR products. The primary disadvantage of this detection method is that it detects accumulation of both specific and nonspecific PCR products.

Real-time systems for PCR were improved by the introduction of fluorogeniclabeled probes that use the 5′ nuclease activity of Taq DNA polymerase. The availability of these fluorogenic probes enabled the development of a real-time method for detecting only specific amplification products.

How the TaqMan

Reagents Work

TaqMan reagents use a fluorogenic probe to detect a specific PCR product as it accumulates during the PCR. Here is how it works:

Step 1 – An oligonucleotide probe is constructed with a fluorescent reporter dye bound to the 5′ end and a quencher on the 3′ end.

Step 2 – While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET; Förster resonance) (Förster, V. T. 1948) through space.

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Chapter 2 Reagent Overview

STRAND DISPLACEMENT

CLEAVAGE

R = REPORTER

Q = QUENCHER

POLYMERIZATION

PROBE

REVERSE PRIMER

FORWARD

PRIMER

POLYMERIZATION COMPLETED

Step 1: A reporter (R) and a quencher (Q) are attached to the 5

′ and 3′

ends of a TaqMan

probe.

Step 3: During each extension cycle, Taq DNA polymerase cleaves the reporter dye from the probe.

Step 4: Once separated from the quencher, the reporter dye emits its characteristic fluorescence.

Step 2: When both labels are attached to the probe, reporter dye emission is quenched.

Step 3 – If the target is present, the probe anneals between primer sites and is cleaved by the 5′ nuclease activity of Taq DNA polymerase during extension. Cleavage of the probe:

• Separates the reporter dye from the quencher, increasing the reporter dye signal.

• Removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process.

Step 4 – More reporter dye molecules are cleaved from their respective probes with each cycle, resulting in an increase in fluorescence intensity proportional to the quantity of amplicon produced. The higher the starting copy number of the nucleic acid target, the earlier a significant increase in fluorescence is observed.

Figure 2-1 illustrates this process.

Taq Ma n® Probes

Figure 2-1 How the TaqMan reagents work

Applied Biosystems offers two types of TaqMan® probes:

•TaqMan

MGB (minor groove-binder) probes with non fluorescent quencher

(NFQ)

•TaqMan

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA dye as a

probes with TAMRA™ dye as quencher

reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

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Page 28

Chapter 2 Reagent Overview

Applied Biosystems offers the following TaqMan probes for use on the StepOne system or the StepOnePlus system:

Probe Assays 5′ Label Dye 3′ Label Dye MGB? System

Ta qM an probe

Ta qM an® MGB probe

Custom TaqMan® MGB Probe

Custom TaqMan® Probe

MGB

Ta q Ma n® Gene Expression Assays

Ta q Ma n® SNP Genotyping Assays

Custom TaqMan® Gene Expression Assays

Custom TaqMan® SNP Genotyping Assays

Custom assays FAM™, TET™,

Custom assays FAM™, TET™ or

TaqMan MGB Probes Recommended

FAM™ dye NFQ Yes StepOne and

StepOne Plus

FAM™ and VIC® dyes

FAM™ dye

FAM™and VIC® dyes

NED™, or VIC® dye

dye

VIC

TAM RA dye

™

No StepOnePlus

systems

system

Applied Biosystems recommends the general use of TaqMan MGB probes, especially when conventional TaqMan probes exceed 30 nucleotides. The TaqMan MGB probes contain:

• A fluorescent reporter dye at the 5′ end – Generates a signal when cleaved by the 5′ nuclease activity of Taq DNA polymerase.

• A nonfluorescent quencher (NFQ) at the 3′ end – Allows Real-Time PCR Systems to measure the reporter dye contributions more precisely than probes with TAMRA dye because the quencher does not fluoresce.

• A minor groove binder (MGB) at the 3′ end – Increases the melting temperature (Tm) of probes without increasing probe length (Afonina et al., 1997; Kutyavin et al., 1997), thereby allowing the design of shorter probes. Consequently, the TaqMan MGB probes exhibit greater differences in Tm values between matched and mismatched probes; greater differences in Tm values provide accurate genotyping.

SYBR® Green Reagents

Experiment

Ty pe s

SYBR® Green reagents include primers and master mixes that contain SYBR® Green dye. You can use SYBR Green reagents for quantitation experiments:

• Standard curve

• Relative standard curve

• Comparative C

Note: You cannot perform multiplex PCR using SYBR Green reagents. For more

information, see “Select Singleplex or Multiplex PCR” on page 3-10.

(ΔΔCT)

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Chapter 2 Reagent Overview

Development of

SYBR Green

Reagents

How the SYBR

Green Reagents

Work

Small molecules that bind to double-stranded DNA can be divided into two classes: those that intercalate DNA and those that bind the minor groove of DNA. Higuchi (Higuchi et al., 1992) used the intercalator ethidium bromide for real-time detection of PCR. Hoechst 33258 is an example of a minor groove-binding dye whose fluorescence increases when bound to double-stranded DNA (Higuchi et al., 1993).

Regardless of the binding method, there are at least two requirements for a DNA binding dye for real-time detection of PCR products:

• Increased fluorescence when bound to double-stranded DNA

• No inhibition of PCR

Applied Biosystems has developed conditions that permit the use of the SYBR

Green I dye in PCR without PCR inhibition and with increased sensitivity of detection compared with ethidium bromide.

The SYBR Green reagents use the SYBR Green I dye to detect PCR products by binding to double-stranded DNA formed during the PCR. Here is how it works:

Step 1 – When added to a sample, SYBR Green I dye immediately binds to all double-stranded DNA.

Step 2 – During the PCR, AmpliTaq Gold

DNA Polymerase amplifies the target, which creates the PCR product, or “amplicon.” The double-stranded DNA is denatured to single-stranded molecules and the SYBR Green I dye is released.

Step 3 – The primers anneal to the single-stranded DNA and the Amplitaq Gold DNA Polymerase amplifies the target, creating more double stranded DNA. As the PCR progresses, more amplicon is created.

Step 4 – The SYBR Green I dye then binds to each new copy of double-stranded DNA that is generated during each PCR cycle. Since the SYBR Green I dye binds to all double-stranded DNA, the result is an increase in fluorescence intensity proportional to the quantity of double-stranded PCR product produced.

Figures 2-2 below illustrates this process.

Step 1: Reaction setup

The SYBR® Green I dye fluoresces when bound to double-stranded DNA.

Step 2: Denaturation

When the DNA is denatured into single-stranded DNA, the

SYBR Green I dye is released and the fluorescence is drastically reduced.

FORWARD

PRIMER

REVERSE

PRIMER

Step 3: Polymerization

During extension, primers anneal and PCR product is generated.

Step 4: Polymerization completed

SYBR® Green I dye binds to the double-stranded product, resulting in a net increase in fluorescence detected by the instrument.

Figure 2-2 How the SYBR Green reagents work

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Chapter 2 Reagent Overview

Selecting the Appropriate Reagent Type

The TaqMan and SYBR Green reagents can be used for the experiment types listed below.

Experiment Type

Reagent Type

SYBR

Green reagents Yes Not

Ta q Ma n

‡ Includes standard curve, relative standard curve, and comparative C

reagents Yes Yes Yes

Quantitation

(Chapter 3)

‡

Genotyping

(Chapter 4)

recommended

Presence/

Absence

(Chapter 5)

recommended

experiments.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

Considerations

for Quantitation

Quantitation experiments can be performed with either TaqMan or SYBR Green reagents. Consider the following when choosing between the two reagent types:

Experiments

Reagent Type Description Advantage Limitation

Ta q Ma n® reagents

Green

SYBR reagents

TaqMan reagents use a fluorogenic probe to enable detection of a specific PCR product as it accumulates during PCR cycles.

SYBR Green reagents use SYBR® Green I dye, a doublestranded DNA binding dye, to detect PCR products as they accumulate during PCR cycles.

• Provides increased specificity with the addition of a fluorogenic probe.

• Provides multiplex capability.

• Includes preformulated assays, optimized to run under universal thermal cycling conditions.

• Can be used for either 1- or 2-step RT-PCR.

• Is economical (no probe needed).

• Allows for melt curve analysis to measure the Tm of all PCR products.

• Can be used for either 1- or 2-step RT-PCR.

Requires synthesis of a unique fluorogenic probe.

Binds nonspecifically to all double-stranded DNA sequences. To avoid false positive signals, check for nonspecific product formation using melt curve or gel analysis.

Not

™

dye

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Page 31

Minimizing DNA Contaminants

The DNA amplification capability of the PCR process makes special laboratory practices necessary when you perform experiments using TaqMan or SYBR Green reagents. Potential contamination can be introduced by samples with high DNA concentrations, either from the DNA template controls or from PCR carryover.

In addition, due to the nonspecific nature of the SYBR Green I dye, any doublestranded DNA will be detected. When using SYBR Green reagents, check for nonspecific product formation by using melt curve or gel analysis. Take care to avoid contamination with target DNA. Gene expression assays that span exon-exon junctions minimize the effect of gDNA (genomic DNA) contaminants.

Chapter 2 Reagent Overview

Using UNG to

Minimize

Reamplification

of Carryover

Products

AmpErase® uracil-N-glycosylase (UNG) is a 26-kDa recombinant enzyme encoded by the Escherichia coli uracil-N-glycosylase gene. This gene has been inserted into an E. coli host to direct expression of the native form of the enzyme (Kwok and Higuchi, 1989).

UNG acts on single- and double-stranded dU-containing DNA. It acts by hydrolyzing uracil-glycosidic bonds at dU-containing DNA sites. The enzyme causes the release of uracil, thereby creating an alkali-sensitive apyrimidic site in the DNA. The enzyme has no activity on RNA or dT-containing DNA (Longo et al.,

1990).

TaqMan Assays

For TaqMan® assays, AmpErase®UNG treatment can prevent the reamplification of carryover PCR products from previous PCR reactions. When dUTP replaces dTTP in PCR amplification, AmpErase UNG treatment can remove up to 200,000 copies of amplicon per 50-μL reaction.

Note: AmpErase UNG (also abbreviated as UDG for uracil-DNA-glycosylase) is

included in some Applied Biosystems TaqMan master mix formulations and can also be purchased individually. When purchasing TaqMan master mixes, check the product information to see if the master mix contains AmpErase UNG.

SYBR Green I Dye Assays

For SYBR Green I dye assays, AmpErase UNG treatment can prevent the reamplification of carryover PCR products from previous PCR reactions. Although Powe r SYBR

Green PCR Master Mix and SYBR® Green PCR Master Mix do not contain AmpErase UNG, they contain dUTP and are thus compatible with AmpErase UNG. If contamination from PCR carryover is suspected, use AmpErase UNG to troubleshoot the problem.

Note: AmpErase UNG can be purchased individually or as part of the

SYBR

Green PCR Core Reagents kit.

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Chapter 2 Reagent Overview

General PCR

Practices

Use the following precautions to minimize sample contamination and PCR product carryover:

• Wear a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation) and clean gloves when preparing samples for PCR amplification. Change gloves whenever you suspect that they are contaminated.

• Maintain separate areas, dedicated equipment, and supplies for: – Sample preparation. – PCR setup. Never bring amplified PCR products into the PCR setup area. – PCR amplification. – Analysis of PCR products.

• Open and close all sample tubes carefully. Avoid splashing or spraying PCR samples.

• Use positive-displacement or air-displacement pipettors with filter-plugged tips. Change tips after each use.

• Keep reactions and components capped as much as possible.

• Clean lab benches and equipment periodically with 10% bleach solution or 70% ethanol.

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Page 33

Quantitation Experiments 3

This chapter covers:

Section 3.1 About Quantitation Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Section 3.2 Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15

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Page 34

Chapter 3 Quantitation Experiments

3-2 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

Page 35

Chapter 3 Quantitation Experiments

Section 3.1 About Quantitation Experiments

This section covers:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Select a Quantitation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5

Select 1-Step or 2-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8

Select Singleplex or Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10

Select the Reagent Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-12

Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-12

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Chapter 3 Quantitation Experiments

Overview

What Is a

Quantitation

Experiment?

How Quantitation

Experiments

Work

A quantitation experiment is a real-time experiment that measures the quantity of a target nucleic acid sequence (target) during each amplification cycle of the polymerase chain reaction (PCR). The target can be DNA, cDNA, or RNA.

Three types of quantitation experiments are discussed in this guide:

• Standard curve (page 3-5)

• Relative standard curve (page 3-5)

• Comparative C

In real-time quantitation experiments, the reactions are characterized by the point in time during cycling when amplification of a PCR product achieves a fixed level of fluorescence, rather than the final quantity of PCR product accumulated after a fixed number of cycles. An amplification plot graphically displays the fluorescence detected over the number of cycles that were performed.

In the initial cycles of PCR, there is no significant change in fluorescence signal. This predefined range of PCR cycles is called the baseline. First, the software generates a baseline-subtracted amplification plot by calculating a mathematical trend of the normalized fluorescent reporter signal (Rn values corresponding to the baseline cycles). Then, an algorithm searches for the point on the amplification plot at which the baseline-corrected normalized fluorescent reporter signal (delta Rn [ΔRn] value) crosses a set threshold. The cycle at which the ΔRn value crosses the threshold is defined as the C

(ΔΔCT) (page 3-6)

Workflow

Before performing quantitation experiments on the Applied Biosystems StepOne and StepOnePlus

1. Select a quantitation method (page 3-5).

2. Select 1- or 2-step RT-PCR (page 3-8).

3. Select singleplex or multiplex PCR reactions (page 3-10).

4. Select the reagent type (page 3-12).

5. Select the assay type (page 3-12).

6. Review the design guidelines for the assay type you selected (Section 3.2 on

page 3-15).

™

Real-Time PCR Systems, prepare for the experiment as follows:

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Select a Quantitation Method

Chapter 3 Quantitation Experiments

About Standard

Curve

Experiments

About Relative

Standard Curve

Experiments

The standard curve method is used to determine the absolute target quantity in samples. With the standard curve method, the StepOne software measures amplification of the target in samples and in a standard dilution series. Data from the standard dilution series are used to generate the standard curve. Using the standard curve, the software interpolates the absolute quantity of target in the samples.

Components

The following components are required when setting up PCR reactions for standard curve experiments:

• Sample – The sample in which the quantity of the target is unknown.

• Standard – A sample that contains known standard quantities; used in quantitation experiments to generate standard curves.

• Standard dilution series – A set of standards containing a range of known quantities. The standard dilution series is prepared by serially diluting standards.

• Replicates – The total number of identical reactions containing identical samples, components, and volumes.

• Negative Controls – Wells that contain water or buffer instead of sample template. No amplification of the target should occur in negative control wells.

The relative standard curve method is used to determine relative target quantity in samples. With the relative standard curve method, the StepOne software measures amplification of the target and of the endogenous control in samples, in a reference sample, and in a standard dilution series. Measurements are normalized using the endogenous control. Data from the standard dilution series are used to generate the standard curve. Using the standard curve, the software interpolates target quantity in the samples and in the reference sample. The software determines the relative quantity of target in each sample by comparing target quantity in each sample to target quantity in the reference sample.

Relative standard curve experiments are commonly used to:

• Compare expression levels of a gene in different tissues.

• Compare expression levels of a gene in a treated sample vs. an untreated sample.

• Compare expression levels of wild-type alleles vs. mutated alleles.

Components

The following components are required when setting up PCR reactions for relative standard curve experiments:

• Sample – The sample in which the quantity of the target is unknown.

• Reference sample – The sample used as the basis for relative quantitation results. For example, in a study of drug effects on gene expression, an untreated control would be an appropriate reference sample. Also called calibrator.

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Chapter 3 Quantitation Experiments

• Standard – A sample that contains known standard quantities; used in quantitation experiments to generate standard curves.

• Standard dilution series – A set of standards containing a range of known quantities. The standard dilution series is prepared by serially diluting standards.

• Endogenous control – A target or gene that should be expressed at similar levels in all samples you are testing. The endogenous control is used to normalize fluorescence signals for the target you are quantifying. Housekeeping genes can be used as endogenous controls.

• Replicates – The total number of identical reactions containing identical samples, components, and volumes.

• Negative Controls – Wells that contain water or buffer instead of sample template. No amplification of the target should occur in negative control wells.

About

Comparative C

Experiments

The comparative CT (ΔΔCT) method is used to determine the relative target quantity in samples. With the comparative C

method, the StepOne software measures

amplification of the target and of the endogenous control in samples and in a reference sample. Measurements are normalized using the endogenous control. The software determines the relative quantity of target in each sample by comparing normalized target quantity in each sample to normalized target quantity in the reference sample.

Comparative C

experiments are commonly used to:

• Compare expression levels of a gene in different tissues.

• Compare expression levels of a gene in a treated sample vs. an untreated sample.

• Compare expression levels of wild-type alleles vs. mutated alleles.

Components

The following components are required when setting up PCR reactions for comparative C

experiments:

• Sample – The sample in which the quantity of the target is unknown.

• Replicates – The total number of identical reactions containing identical samples, components, and volumes.

• Negative Controls – Wells that contain water or buffer instead of sample template. No amplification of the target should occur in negative control wells.

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Chapter 3 Quantitation Experiments

Comparison of

Quantitation

Consider the following when choosing between standard curve, relative standard curve, and comparative C

Methods

Experiment

Ty pe

Standard curve Uses a standard curve to

Relative standard curve

Comparative

(ΔΔCT)

determine the absolute quantity of a target in a sample. Typically used for quantifying viral load.

Uses a standard curve to determine the change in expression of a target in a sample relative to the same target in a reference sample. Best for assays that have suboptimal PCR efficiency.

Uses arithmetic formulas to determine the change in expression of a target in a sample relative to the same target in a reference sample. Best for high-throughput measurements of relative gene expression of many genes in many samples.

Description Advantage Limitation

experiments:

Allows comparisons against known standard quantities.

Requires the least amount of validation because the PCR efficiencies of the target and endogenous control do not need to be equivalent.

• Relative levels of target in samples can be determined without the use of a standard curve, provided that the PCR efficiencies of the target and endogenous control are relatively equivalent.

• Reduced reagent usage.

• More space is available in the reaction plate.

Because a standard curve must be constructed for each target, standard curve experiments require more reagents and more space in the reaction plate.

Because a standard curve must be constructed for each target, relative standard curve experiments require more reagents and more space in the reaction plate.

• Suboptimal (low PCR efficiency) assays may produce inaccurate results.

• Before you use the comparative C Applied Biosystems recommends that you determine that the PCR efficiencies for the target assay and the endogenous control assay are approximately equal.

method,

For More

Information

For more information on quantitation methods, refer to User Bulletin #2: Relative Quantitation of Gene Expression.

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Chapter 3 Quantitation Experiments

Single Tu be

Select 1-Step or 2-Step RT-PCR

Reverse transcription-polymerase chain reaction (RT-PCR) is used to quantify RNA. RT-PCR can be performed as a 1-step or 2-step procedure.

About 1-Step

RT-PCR

In 1-step RT-PCR, you perform reverse transcription and the PCR in a single buffer system (Figure 3-1). The reaction proceeds without the addition of reagents between the RT and PCR steps. 1-step RT-PCR offers the convenience of a single-tube preparation for RT and PCR amplification. However, the carryover prevention enzyme, AmpErase

uracil-N-glycosylase (UNG), cannot be used with 1-step RTPCR. In 1-step RT-PCR, the presence of UNG would destroy the cDNA as it is being made. For information about UNG, see “Using UNG to Minimize Reamplif ication of

Carryover Products” on page 2-7.

Figure 3-1 Schematic representation of 1-step RT-PCR

About 2-Step

RT-PCR

In 2-step RT-PCR, you perform two separate reactions: one for RT and one for PCR (Figure 3-2). 2-step RT-PCR is useful when detecting multiple transcripts from a single cDNA reaction, or when storing a portion of the cDNA for later use. When you perform PCR using dUTP as one of the bases, you can use AmpErase

UNG enzyme to prevent carryover contamination. For information about UNG, see “Using

UNG to Minimize Reamplification of Carryover Products” on page 2-7.

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Chapter 3 Quantitation Experiments

Tube 1

Tube 2

PCR Step

Oligo d(T) or random hexamer

Figure 3-2 Schematic representation of 2-step RT-PCR

Primers Used for

cDNA Synthesis

For 1-step RT-PCR, sequence-specific reverse primers can be used for cDNA synthesis.

For 2-step RT-PCR, the following primers can be used for cDNA synthesis:

• Oligo d(T)

• Random primers

• Sequence-specific reverse primers

The choice of primers for reverse transcription is best made after experimentally evaluating all three priming systems. For short RNA sequences containing no hairpin loops, any of the three priming systems works equally well. For longer RNA transcripts or sequences containing hairpin loops, consider the following guidelines:.

Primers Selection Guidelines

Oligo d(T)

Random primers • Try first for use with long reverse transcripts or reverse

Sequence-specific reverse primers

• Use to reverse transcribe only eukaryotic mRNAs and retroviruses with poly-A tails

• Avoid long mRNA transcripts or amplicons greater than 2 kilobases upstream from the poly-A site

transcripts containing hairpin loops

• Use to transcribe all RNA (rRNA, mRNA, and tRNA)

• Use to reverse transcribe RNA-containing complementary sequences only

•Use in 1-step RT-PCR

Comparison of

RT-PCR Methods

Method Primers for cDNA Synthesis Comments

1-step RT-PCR

2-step RT-PCR

Sequence-specific reverse primer Requires single reaction mix

AmpErase

Random hexamers cDNA can be stored for later use

Oligo d(T)

Sequence-specific reverse primers

AmpErase

Requires two reaction mixes

UNG cannot be used

UNG can be used

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Chapter 3 Quantitation Experiments

Select Singleplex or Multiplex PCR

You can perform a PCR reaction using either:

• Singleplex PCR – In singleplex PCR a single primer set is present in the reaction tube or well. Only one target or endogenous control can be amplified per reaction.

• Multiplex PCR – In multiplex PCR, two or more primer sets are present in the reaction tube or well. Each set amplifies a specific target or endogenous control. Typically, a probe labeled with FAM labeled with VIC

IMPORTANT! SYBR

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

Singleplex PCR

dye detects the endogenous control.

Green reagents cannot be used for multiplex PCR.

Multiplex PCR

™

dye detects the target and a probe

Target Primer Set

Endogenous Control

Endogenous Control Primer Set

Primer Set

cDNA

GR2331

™

About Multiplex

PCR

Figure 3-3 Singleplex vs. multiplex PCR

In order to perform multiplex PCR you must:

• Ensure that the endogenous control you have selected is more abundant (lower

value) than all of the targets that you are trying to quantify under all

conditions.

• Run the endogenous control assay as a primer-limited assay. The endogenous control assay (for the more abundant template) in each reaction must be primerlimited to avoid competitive PCR that may alter the C

of the less abundant

template.

Primer Limiting in Multiplex PCR

To generate an accurate multiplex assay, it is important that the amplification of one species does not dominate amplification of the other species. Otherwise, the amplification of a highly abundant species can prevent the less-abundant species from amplifying eff iciently.

If the less-abundant species does not amplify efficiently, your experiment may produce inaccurate results or, in severe cases, detection of the less-abundant species may be inhibited completely. You can avoid this situation by limiting the concentrations of the primers used to amplify the more abundant species, thereby “turning off” the amplification soon after the C

has been established. However, a

primer-limited assay may be more susceptible to fluctuations in reaction conditions than the primer non-limited target assay that it is normalizing. For more information, see Appendix B, “Primer Limiting in Multiplex PCR.”

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Chapter 3 Quantitation Experiments

Singleplex vs.

Multiplex PCR

Primer limiting in multiplex PCR becomes increasingly more complex as the number of targets you quantify increases. When you analyze multiple numbers of targets, it may be more effective to use singleplex PCR for the following reasons:

• In multiplex PCR, it may be difficult to find a suitable endogenous control, one that:

– Is more abundant than all of the targets you are quantifying. – Does not change expression levels with experimental conditions or across

different samples.

You would first have to run all of your target assays and endogenous control assays in both the multiplex and singleplex formats, then compare C

from both formats to determine if there are any effects of the multiplexing on your C

values.

• In singleplex PCR, any target that does not change expression levels with experimental conditions or across samples may serve as an endogenous control. Therefore, the more targets you have in a singleplex format, the higher the probability that you will have one or more suitable endogenous controls against which to normalize your remaining targets.

Consider the following when choosing between multiplex and singleplex PCR:

PCR Description Advantage Limitation

Singleplex A reaction in which a

single target or endogenous control is amplified in the reaction tube or well.

• No optimization is required for Ta qM an

• Any target that does not change expression levels with experimental conditions or across samples may serve as an endogenous control.

• Flexibility to use TaqMan SYBR® Green reagents.

assays.

• Requires sample for both the target and the endogenous control.

values

Multiplex A reaction in which

more than one target or endogenous control is amplified in the reaction tube or well.

• Reduces both the running costs and the dependence on accurate pipetting when splitting a sample into two separate tubes.

• The endogenous control assay must be run as a primer-limited assay.

• Requires validation and optimization.

• May not be as effective as singleplex PCR when analyzing multiple numbers of targets.

• You cannot use SYBR reagents.

Green

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Chapter 3 Quantitation Experiments

Select the Reagent Type

TaqMan vs.

SYBR Green

Reagents

You can perform quantitation experiments on the StepOne and StepOnePlus systems with:

•TaqMan

• SYBR

For information on choosing between TaqMan and SYBR Green reagents, see

“Considerations for Quantitation Experiments” on page 2-6.

Green reagents

Select the Assay Type

When you design your experiments with the StepOne software, you can select the following assay types for quantitation experiments:

• Inventoried/Made to Order (page 3-13)

•Custom (page 3-14)

This section lists the products available for each assay type.

Note: The assays are specific to the target of interest. The master mixes contain the

remaining components needed for the PCR reaction.

reagents

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Chapter 3 Quantitation Experiments

Inventoried/Made

to Order Assays

Product Attributes

Ta q Ma n

Gene

Expression Assays

Ta q Ma n

Endogenous

Control Assays

Note: FAM

™

dyelabeled TaqMan® Endogenous Control Assays are included as Inventoried TaqMan Gene Expression Assays.

Custom TaqMan

Gene

Expression Assays

Ta q Ma n

Gene

Expression Master Mix

Ta q Ma n

2✕ Universal PCR Master Mix (with or without AmpErase

Ta q Ma n PCR Master Mix (2✕), No AmpErase

UNG)

Fast Universal

UNG

• Predesigned, gene-specific primer and probe sets for human, mouse, rat, Arabidopsis, Drosophila, C. elegans, C. familiares (dog), and M. mulatta (Rhesus) genes.

™

•Probe is a FAM

dye-labeled MGB probe.

• Provided in a convenient, single, 20✕ tube.

• Available as Inventoried or Made to Order assays.

• Optimized, preformulated, ready-to-use endogenous control assays.

• Cost-effective gene expression quantitation for human, mouse, rat, Arabidopsis, Drosophila, and any eukaryotic species.

™

•Choice of FAM

dye or VIC® dye labels (primer-limited).

• Any species or organism.

• Target of your choice.

•Probe is a FAM™ dye-labeled MGB probe.

• Provided in a convenient, single, 205 tube.

Tailored for precise quantitation by real-time PCR for routine and challenging experiments:

• Sensitive detection down to 1 copy of target.

• Multiplex PCR for co-amplifying two targets in a single reaction.

• Specificity for differentiation between gene family members.

• Validated with TaqMan

Gene Expression Assays.

• Simplifies assay implementation by using one reagent for all assays.

• Provides optimal performance for TaqMan

assays that

use cDNA or DNA as a template.

• Contains components that ensure excellent assay performance.

• The use of one reagent for all assays simplifies the process of assay implementation.

• Provides the same attributes listed above for TaqMan

2✕

Universal PCR Master Mix.

• Allows you to run quantitation experiments on the StepOne

™

and StepOnePlus™ systems in about 35 min.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

For guidelines on designing your experiments with Inventoried/Made to Order assays, see page 3-16.

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Chapter 3 Quantitation Experiments

Custom Assays

Product Attributes

Custom TaqMan

Probes

and Primers

Primer Express

Software

Ta q Ma n

Gene

Expression Master Mix

Ta q Ma n

2✕ Universal PCR Master Mix (with or without AmpErase

Ta q Ma n PCR Master Mix (2✕), No AmpErase

Power SYBR

UNG)

Fast Universal

UNG

Green

PCR Master Mix

Green PCR

SYBR Master Mix

• Any species or organism.

• Choice of dye labels, quenchers, and synthesis scales.

• For use with the Primer Express® Software and Applied Biosystems Assay Design Guidelines.

Software that designs primers and probes for real-time PCR.

Tailored for precise quantitation by real-time PCR for routine and challenging experiments:

• Sensitive detection down to 1 copy of target.

• Multiplex PCR for co-amplifying two targets in a single reaction.

• Specificity for differentiation between gene family members.

• Validated with TaqMan

Gene Expression Assays.

• Simplifies assay implementation by using one reagent for all assays.

• Provides optimal performance for TaqMan

assays that

use cDNA or DNA as a template.

• Contains components that ensure excellent assay performance.

• The use of one reagent for all assays simplifies the process of assay implementation.

• Provides the same attributes listed above for TaqMan

2✕

Universal PCR Master Mix.

• Allows you to run quantitation experiments on the StepOne

™

and StepOnePlus™ systems in about 35 min.

• Highly sensitive quantitation enables low-copy-number detection (as few as two copies of a target gene).

• Detects double-stranded DNA, so specific probes are not required.

• Contains highly purified AmpliTaq Gold

DNA polymerase LD to minimize nonspecific product formation (including primer-dimer).

• For use with the Primer Express

Software and Applied

Biosystems Assay Design Guidelines.

• Detects double-stranded DNA, so specific probes are not required.

• For standard applications when high sensitivity is not required.

• Contains AmpliTaq Gold

DNA polymerase to minimize

nonspecific product formation (including primer-dimer).

• For use with the Primer Express

Software and Applied

Biosystems Assay Design Guidelines.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

For guidelines on designing your experiments with Custom assays, see page 3-21.

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Section 3.2 Design Guidelines

This section covers:

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16

TaqMa n Custom TaqMan

Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19

Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21

Design Primers and Probes Using Primer Express

Select the Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24

Use the Recommended Thermal Cycling Conditions . . . . . . . . . . . . . . . . . 3-27

Optimize Primer Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-30

Optimize the Probe Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-33

For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-35

Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16

Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . 3-18

Chapter 3 Quantitation Experiments

Software . . . . . . . . . . 3-21

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Chapter 3 Quantitation Experiments

Inventoried/Made to Order Assays

Workflow

If you select the Inventoried/Made to Order assay type in the StepOne™ software, Applied Biosystems recommends that you follow the workflow below:

1. Select the assay:

•TaqMan

Gene Expression Assays (below).

• Custom TaqMan

2. Select the master mix (page 3-19).

3. Design the experiment using the StepOne software (page 3-20).

TaqM an® Gene Expression Assays

Product

Description

TaqMan® Gene Expression Assays are a comprehensive collection of Inventoried and Made to Order probe and primer sets for performing quantitation experiments on human, mouse, rat, Arabidopsis, Drosophila, C. elegans, C. familiares (dog), and M. mulatta (Rhesus) genes.

The assays:

•Use TaqMan

reagents to amplify and detect the target in cDNA samples.

• Are designed using an automated design and quality-controlled pipeline. Inventoried assays are manufactured and placed in inventory; Made to Order assays are predesigned and manufactured when ordered.

• Are designed and optimized to work with an Applied Biosystems TaqMan master mix, using universal thermal cycling conditions.

• When possible, amplify target cDNA without amplifying genomic DNA (m suffix in assay ID) by designing probes that cross exon-exon junctions.

Gene Expression Assays (page 3-18).

Product

Requirements

All TaqMan Gene Expression Assays require:

• Three components: – 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells

in a study having the same amount of cDNA.

–20✕ Gene Expression Assay Mix (specific for each target). Each assay mix

consists of two unlabeled PCR primers and a FAM

™

dye-labeled TaqMan® MGB (minor groove binder) probe in a preformulated 20✕ mix. 1✕ final concentrations are 250 nM for the probe and 900 nM for each primer.

–TaqMan

Mix (with or without AmpErase Master Mix, No AmpErase

Gene Expression Master Mix, TaqMan® Universal PCR Master

UNG), or TaqMan® Fast Universal PCR

UNG.

• Only one PCR amplification step during each PCR cycle and a simultaneous real-time reading to obtain results.

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Chapter 3 Quantitation Experiments

Available Assays

TaqMan Gene Expression Assays are available for human, mouse, rat, Arabidopsis, Drosophila, C. elegans, C. familiares (dog), and M. mulatta (Rhesus) genes. The part

numbers are:

• PN 4331182 for Inventoried assays

• PN 4351372 for Made to Order assays

The prefix of the assay name indicates the species for which the assay was designed:

Hs for Homo sapiens (human), Mm for Mus musculus (mouse), Rn for Rattus norvegicus (rat), At for Arabidopsis thaliana, Dm for Drosophila melanogaster,

Ce for C. elegans, Cf for C. familiares (dog), and Rh for M. mulatta (Rhesus).

The suffix of the assay name indicates the assay placement, as described in the table below.

Suffix Description

_m The assay’s probe spans an exon junction; the assay does not detect

genomic DNA.

_s The assay’s primers and probes are designed within a single exon; the assay

does detect genomic DNA.

_g The assay may detect genomic DNA; the assay’s primers and probes may be

within a single exon.

_mH The assay was designed to a transcript belonging to a gene family with high

_sH

_gH

_u The assay’s amplicon spans an exon junction and the probe sits completely in

sequence homology. The assay provides between 10 C between the target gene and the gene with the closest sequence homology. Therefore, the assay detects the target transcript with 1000- to 30,000-fold greater discrimination (sensitivity) than the closest homologous transcript, if they are present at the same copy number in a sample.

one of the spanned exons.

and 15 CT difference

Taq Ma n® Endogenous Control Assays

TaqMa n® Endogenous Control Assays are available as:

• Inventoried TaqMan Gene Expression Assays (PN 4331182) – Each assay

™

contains a FAM

dye-labeled TaqMan® MGB probe in a single, preformulated

20✕ tube.

• Individual control assays for all human, mouse, and rat species (various part numbers) – Each assay contains either a FAM probe, a VIC

dye-labeled TaqMan® MGB probe, or a TAMRA™ dye-labeled

™

dye-labeled TaqMan® MGB

probe. TaqMan Endogenous Controls with VIC dye labels are primer-limited.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

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Chapter 3 Quantitation Experiments

For More

Information

• For information on the latest available products and specific product uses, refer to the Applied Biosystems Web site:

http://www.appliedbiosystems.com/

a. On the Home page, under TaqMan

Products, select Taq Ma n® Gene

Expression Assays.

b. On the Gene Expression Assays & Arrays page:

• Under Individual Assays, select Taq Ma n

Gene Expression Assays.

This option links to all TaqMan Gene Expression Assays, including TaqMan Endogenous Control Assays that contain FAM dye-labeled probes.

• Under Individual Control Assays, select Ta qM a n

Endogenous

Control Assays. This option links to the individual TaqMan

Endogenous Control Assays (that contain FAM dye-labeled TaqMan MGB probes, VIC dye-labeled TaqMan MGB probes, or TAMRA dye-labeled probes).

• For information on Custom TaqMan Endogenous Control Assays, refer to the

Using TaqMan

Endogenous Control Assays to Select an Endogenous Control

for Experimental Studies Application Note.

• For information on preparing PCR reactions using the TaqMan Gene Expression Assays, refer to the TaqMan

Gene Expression Assays Protocol.

Custom TaqMan® Gene Expression Assays

Product

Description

Product

Requirements

Custom TaqMan® Gene Expression Assays are TaqMan probe and primer sets that are designed, synthesized, and formulated by the Custom TaqMan service based on sequence information that you submit. Custom TaqMan Gene Expression Assays allow you to perform quantitation experiments on any gene or splice variant in any organism.

The assays:

•Use TaqMan

reagents to amplify and detect the target in cDNA samples.

• Are developed using proprietary assay-design software.

• Are designed and optimized to work with an Applied Biosystems TaqMan master mix, using universal thermal cycling conditions.

All Custom TaqMan Gene Expression Assays require:

• A submission file that includes your target sequence. You create the submission file using free File Builder software, then submit the file to the Custom

Taq Ma n

Genomic Assays service.

• Three components: – 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells

in a study having the same amount of cDNA.

–20✕ Gene Expression Assay or 60✕ Gene Expression Assay (specific for

each target). Each assay consists of two target-specific primers and a FAM dye-labeled TaqMan MGB probe in a preformulated 20✕ or 60✕ mix. 1✕ final concentrations are 250 nM for the probe and 900 nM for each primer.

Genomic Assays

™

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Chapter 3 Quantitation Experiments

• Only one PCR amplification step during each PCR cycle and a simultaneous

For More

• For information on the latest available products and specific product uses, refer

Information

• For information on ordering Custom TaqMan Gene Expression Assays, refer to

• For information on preparing PCR reactions using the Custom TaqMan Gene

Select the Master Mix

–TaqMan® Gene Expression Master Mix, TaqMan® Universal PCR Master

Mix (with or without AmpErase Master Mix, No AmpErase

UNG), or TaqMan® Fast Universal PCR

UNG.

real-time reading to obtain results.

to the Applied Biosystems Web site:

http://www.appliedbiosystems.com/

a. On the Home page, under TaqMan

Products, select TaqM an® Gene

Expression Assays.

b. On the Gene Expression Assays & Arrays page, under Individual Assays,

select Custom TaqMan

the Custom TaqMan

Genomic Assays Protocol: Submission Guidelines.

Expression Assays, refer to the Custom TaqMan

Gene Expression Assays.

Gene Expression Assays

Protocol.

Available Master

Mixes

Applied Biosystems Inventoried/Made to Order assays for quantitation experiments are designed to work with the following TaqMan

Master Mix Part Number

Ta q Ma n 200 reactions

Ta q Ma n 2000 reactions

Ta q Ma n 250 Reactions

Ta q Ma n

10-Pack, TaqMan

Ta q Ma n Reactions

10-Pack, TaqMan UNG

Gene Expression Master Mix, 1-Pack (1 × 5 mL),

Gene Expression Master Mix, 10-Pack (10 × 5 mL),

Fast Universal PCR Master Mix (2✕), No AmpErase® UNG,

2✕ Universal PCR Master Mix, 200 Reactions 4304437

2✕ Universal PCR Master Mix, 2000 Reactions 4326708

2✕ Universal PCR Master Mix 4305719

2✕ Universal PCR Master Mix, No AmpErase® UNG, 200

2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000

2✕ Universal PCR Master Mix, No AmpErase®

master mixes:

4369016

4369542

4352042

4324018

4326614

4324020

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Chapter 3 Quantitation Experiments

For More

Information

For information on using the TaqMan reagents, refer to:

• TaqMan

Design the Experiment

Use the StepOne

Software

For More

Information

For Applied Biosystems Inventoried/Made to Order assays, use the StepOne software to design your quantitation experiments. The StepOne software automatically calculates volumes for the:

• Reaction mix components

• Sample dilutions

• (Standard curve and relative standard curve experiments only) Standard dilution series

Note: To select the Inventoried/Made to Order assay type in the StepOne software,

go to the Reaction Setup screen in either the Design Wizard or Advanced Setup, then select Inventoried/Made to Order from the Assay Type dropdown menu.

For information on designing and performing quantitation experiments on the StepOne and StepOnePlus systems, refer to:

• Applied Biosystems StepOne

Getting Started Guide for Standard Curve Experiments

• Applied Biosystems StepOne Getting Started Guide for Relative Standard Curve and Comparative C Experiments

Fast Universal PCR Master Mix (2✕) Protocol

Gene Expression Master Mix Protocol

Universal PCR Master Mix Protocol

™

and StepOnePlus™ Real-Time PCR Systems

™

and StepOnePlus™ Real-Time PCR Systems

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Custom Assays

Chapter 3 Quantitation Experiments

Workflow

If you select the Custom assay type in the StepOne™ software for a quantitation experiment (that is, you are designing your own primers and probes), Applied Biosystems recommends that you follow the workflow for the Applied Biosystems Assay Design Guidelines:

1. Design primers and probes using Primer Express

Software (below).

2. Select the appropriate reagents (page 3-24).

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

3. Use the recommended thermal cycling conditions (page 3-27).

4. Begin with default primer and probe concentrations. If needed, optimize the primer concentrations (page 3-30) and probe concentrations (page 3-33).

IMPORTANT! These steps provide a rapid and reliable system for assay design and

optimization only when used in their entirety. Adopt the system as a whole to achieve the highest level of success. For a more detailed description of Applied Biosystems Assay Design Guidelines, see Appendix C.

Note: To select the Custom assay type in the StepOne software, go to the Reaction

Setup screen in either the Design Wizard or Advanced Setup, then select Custom from the Assay Type dropdown menu.

Design Primers and Probes Using Primer Express® Software

The Primer Express® Software uses recommended parameters to select primers and probes based on the DNA sequence that you provide.

If you are designing your own assay, follow the summary of the primer and probe design guidelines for quantitation experiments on page 3-23. For a detailed discussion of these guidelines see “About the Primer and Probe Design Guidelines”

on page 3-22.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

Green I dye detection, it is

Selecting an

Amplicon Site for

Quantitation

Assays

Note: Even though a probe is not required for SYBR

still a good idea to use Primer Express software to select a primer and probe set when you design an assay for SYBR

Green reagents. Although no probe will be used, the primers will meet all required criteria; if you need to convert the assay to TaqMan reagents to obtain higher specificity, you can find the probe immediately in the original Primer Express software document.

Selecting a good amplicon site ensures amplification of the target mRNA/cDNA without co-amplifying the genomic sequence, pseudogenes, and other related genes. SYBR Green reagents can be useful for screening amplicon sites for gene expression.

™

dye

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Chapter 3 Quantitation Experiments

Guidelines

• The amplicon should span one or more introns to avoid amplification of the target gene in genomic DNA.

• The primer pair should be specific to the target gene to avoid amplification of pseudogenes or other related genes.

• When designing primers, use Primer Express software guidelines.

• If no good sequence is found, it may be necessary to examine the sequence and redesign the amplicon or simply screen for more sites.

If the gene you are studying does not have introns, it is not possible to design an amplicon that will amplify the mRNA sequence without amplifying the genomic sequence. In this case, run a control of your RNA sample that has not been reversetranscribed (RT minus controls).

About the Primer

and Probe Design

Guidelines

Selection of Small Amplicons

An important default parameter in Primer Express software is the selection of amplicons in the 50- to 150-basepair range. Small amplicons are favored because they promote high-efficiency amplification.

In addition, high-efficiency assays enable relative quantitation to be performed using the comparative C

(ΔΔCT) method (Livak and Schmittgen, 2001). This method

increases sample throughput by eliminating the need for standard curves.

G/C Content

Whenever possible, select primers and probes in a region with a G/C content of 30 to 80%. Regions with a G/C content >80% may not denature well during thermal cycling, leading to a less efficient reaction. G/C-rich sequences are susceptible to nonspecific interactions that may reduce reaction efficiency and produce nonspecific signal in assays using SYBR Green reagents. Avoid primer and probe sequences containing runs of four or more G bases.

Melting Temperature

When you select primers and probes with the recommended melting temperature (Tm), you can use universal thermal cycling conditions. Applied Biosystems recommends that the probe Tm be 10 °C higher than that of the primers.

5′ End of Probes

Primer Express software does not select probes with a G on the 5′ end. The quenching effect of a G base in this position will be present even after probe cleavage. The presence of a G base can result in reduced fluorescence values (ΔRn) that can negatively affect assay performance. G bases in positions close to the 5′ end, but not on it, have not been shown to compromise assay performance.

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Chapter 3 Quantitation Experiments

3′ End of Primers

To reduce the possibility of nonspecific product formation, ensure that the last five bases on the 3′ end of the primers do not contain more than two C and/or G bases. Under certain circumstances, such as a G/C-rich template sequence, you may have to relax this recommendation to keep the amplicon under 150 basepairs in length. In general, avoid primer 3′ ends extremely rich in G and/or C bases.

Summary of

Primer and MGB

Probe Design

Guidelines

Probe Guidelines Primer Guidelines

Select the probe first, then design the primers as close as possible to the probe without overlapping the probe (amplicons of 50 to 150 basepairs are strongly recommended).

Keep the G/C content in the 30 to 80% range.

Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs should be avoided.

When using Primer Express Tm should be 68 to 70 °C.

No G on the 5

Make TaqMan possible without being shorter than 13 nucleotides.

′ end. The five nucleotides at the 3′ end should

MGB probes as short as

Software, the

When using Primer Express® Software, the Tm should be 58 to 60 °C.

have no more than two G and/or C bases.

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Chapter 3 Quantitation Experiments

Select the Reagents

Several TaqMan and SYBR Green reagents are available for quantitation experiments. The reagents you use depend on your target:

• DNA or cDNA (below).

• RNA using 1-step RT-PCR (page 3-25).

• RNA using 2-step RT-PCR (page 3-26).

Note: If you are using SYBR Green reagents, Applied Biosystems highly

recommends the Power SYBR Green reagents.

DNA or cDNA

Quantitation

Reagent Kit Part Number

Ta q Ma n® reagents TaqMan® Gene Expression Master Mix, 1-Pack

(1 × 5 mL), 200 reactions

Ta qM an

Gene Expression Master Mix, 10-Pack

(10 × 5 mL), 2000 reactions

Ta qM an AmpErase

Ta qM an

Fast Universal PCR Master Mix (2✕), No

UNG, 250 Reactions

2✕ Universal PCR Master Mix, 200

Reactions

Ta qM an

2✕ Universal PCR Master Mix, 2000

Reactions

10-Pack, TaqMan

Ta qM an

2✕ Universal PCR Master Mix, No

AmpErase

Ta qM an

2✕ Universal PCR Master Mix, No

AmpErase

10-Pack, TaqMan No AmpErase

Ta qM an

PCR Core Reagents Kit, 200 Reactions N808-0228

2✕ Universal PCR Master Mix 4305719

UNG, 200 Reactions

UNG, 2000 Reactions

2✕ Universal PCR Master Mix,

UNG

4369016

4369542

4352042

4304437

4326708

4324018

4326614

4324020

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Chapter 3 Quantitation Experiments

Reagent Kit Part Number

RNA Quantitation

Using 1-Step

RT-PCR

SYBR® Green reagents

Power SYBR® Green PCR Master Mix (1 mL), 40 reactions

Power SYBR

Green PCR Master Mix (5-mL),

200 reactions

Power SYBR

Green PCR Master Mix

(10 × 5-mL), 2000 reactions

Power SYBR

Green PCR Master Mix (50 mL),

2000 reactions

SYBR

Green PCR Master Mix (1-mL),

40 reactions

SYBR

Green PCR Master Mix (5-mL),

200 reactions

SYBR

Green PCR Master Mix (50-mL),

2000 reactions

SYBR

Green PCR Core Reagents, 200 Reactions 4304886

Reagent Kit Part Number

Ta q Ma n® reagents TaqMan® One-Step RT-PCR Master Mix

Reagents Kit

Ta qM an

Note: Use the TaqMan

EZ RT-PCR Core Reagents

EZ RT-PCR Core

N808-0236

Reagents when a high-temperature RT step is required.

SYBR reagents

Green

Power SYBR® Green RT-PCR Reagents Kit 4368711

SYBR

Green RT-PCR Reagents 4310179

4368577

4367659

4368708

4367660

4344463

4309155

4334973

4309169

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Chapter 3 Quantitation Experiments

RNA Quantitation

Using 2-Step

RT-PCR

Reagent Step Kit Part Number

Ta q Ma n reagents

SYBR reagents

Green

PCR step only TaqMan® Gene Expression Master

RT step only High-Capacity cDNA Reverse

Both RT and PCR steps

PCR step only Power SYBR® Green PCR Master

Mix, 1-Pack (1 × 5 mL), 200 reactions

Ta q Ma n Mix, 10-Pack (10 × 5 mL), 2000 reactions

Ta q Ma n® 2✕ Universal PCR Master Mix

Ta q Ma n Mix (2✕), No AmpErase® UNG

Tran s c ri pt i on Ki t

Ta q Ma n Reagents

Ta q Ma n

Mix

SYBR

Gene Expression Master

Fast Universal PCR Master

Reverse Transcription

Gold RT PCR kit N808-232

Green PCR Master Mix 4309155

4369016

4369542

4304437

4352042

4374966

N808-234

4367659

About AmpliTaq

Gold DNA

Polymerase

About

MultiScribe

Reverse

Transcriptase

RT step only High-Capacity cDNA Reverse

Both RT and PCR steps

Tran s c ri pt i on Ki t

Ta q Ma n® Reverse Transcription Reagents

Power SYBR Reagents Kit

SYBR

Green RT-PCR

Green RT-PCR Reagents 4310179

4374966

N808-234

4368711

The use of the hot-start enzyme AmpliTaq Gold® DNA Polymerase is an integral part of Applied Biosystems Assay Design Guidelines for both TaqMan and SYBR Green reagents. AmpliTaq Gold DNA Polymerase ensures a robust reaction and it can dramatically reduce the amount of nonspecific product formation. A further benefit is the simplification of assay setup, which can be performed at room temperature.

Note: The DNA polymerase included in the TaqMan Fast Universal PCR Master

Mix (2✕), No AmpErase UNG, is capable of very fast hot-start PCR. The performance is similar to that of the AmpliTaq Gold DNA Polymerase.

MultiScribe™ Reverse Transcriptase is a recombinant Moloney Murine Leukemia Virus (MuLV) Reverse Transcriptase that reverse transcribes RNA into complimentary DNA (cDNA).

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Use the Recommended Thermal Cycling Conditions

Use the thermal cycling conditions recommended for your sample:

• DNA or cDNA (page 3-28).

• RNA using 1-step RT-PCR (page 3-28).

• RNA using 2-step RT-PCR (page 3-29).

Note: Thermal cycling conditions for Fast reagents differ from thermal cycling

conditions for standard reagents.

Chapter 3 Quantitation Experiments

About the

VeriFlex

Te ch no lo gy

The StepOnePlus instrument contains six independently thermally regulated

™

Ve ri Fl ex

™

blocks to help you optimize your thermal cycling conditions.

If you are running your experiment on a StepOnePlus instrument, you can set a different temperature for one or more of the VeriFlex blocks, creating up to six different zones for samples, or you can set the same temperature for each of the VeriFlex blocks.

For more information on using the VeriFlex sample blocks, access the Help from within the StepOne software by clicking in the toolbar or pressing F1.

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Chapter 3 Quantitation Experiments

DNA or cDNA

Quantitation

For TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, follow these conditions:

Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpErase

Activation

2 min @ 50 °C 20 sec @ 95 °C 1 sec @ 95 °C 20 sec @ 60 °C

‡ Required only if AmpErase® UNG is added to the reactions.

UNG

‡

HOLD HOLD CYCLE

Activation Melt Anneal/Extend

For TaqMan Gene Expression Master Mix, TaqMan 2✕ Universal PCR Master Mix, and Power SYBR Green PCR Master Mix, follow these conditions:

Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpErase

Activation

UNG

‡

AmpliTaq Gold®

DNA Polymerase

Activation

Melt Anneal/Extend

RNA Quantitation

Using 1-Step

RT-PCR

HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

‡ Required only if AmpErase® UNG is added to the reactions or is included in the master mix.

For the TaqMan One-Step RT-PCR Master Mix Reagents Kit and Power SYBR Green RT-PCR Reagents Kit, follow these conditions:

Times and Temperatures

Initial Steps PCR (40 Cycles)

Reverse

Tr an sc r ip ti o n

HOLD HOLD 40 CYCLES

30 min @ 48 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

Note:

TaqMan

These conditions do not apply to the TaqMan EZ RT-PCR Kit. See the

EZ RT-PCR Kit Protocol for the appropriate conditions.

AmpliTaq Gold

DNA Polymerase

Activation

Melt Anneal/Extend

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Chapter 3 Quantitation Experiments

RNA Quantitation

Using 2-Step

RT-PCR

For the High-Capacity cDNA Reverse Transcription Kit, follow these conditions:

Step Times and Temperatures

Step 1 Step 2

1. RT Step

After reverse-transcribing the RNA into cDNA (RT step), samples can be stored or used for the subsequent PCR step described below.

IMPORTANT!

For most applications and when large amounts of cDNA are required,

HOLD HOLD

10 min @ 25 °C 120 min @ 37 °C

Applied Biosystems recommends 120 minutes at 37 °C for reverse transcription to achieve optimal conversion.

For TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, follow these conditions:

Step Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpliTaq

DNA

Gold

Polymerase

Activation

Melt

Anneal/

Extend

2. PCR Step

AmpErase

UNG

Activation

‡

HOLD HOLD CYCLE

2 min @ 50 °C 20 sec @ 95 °C 1 sec @ 95 °C 20 sec @ 60 °C

‡ Required only if AmpErase® UNG is added to the reactions.

For TaqMan Gene Expression Master Mix, TaqMan 2✕ Universal PCR Master Mix (with AmpErase UNG), and Power SYBR Green PCR Master Mix, follow these conditions:

Step Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpliTaq

DNA

Gold

Polymerase

Activation

Melt

Anneal/

Extend

2. PCR Step

AmpErase

UNG

Activation

HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

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Chapter 3 Quantitation Experiments

Optimize Primer Concentrations

By independently varying forward and reverse primer concentrations, you can identify the concentrations that provide optimal assay performance. Primers are always in large molar excess during the exponential phase of PCR amplification.

When you use TaqMan Gene Expression Master Mix or TaqMan 2✕ Universal PCR Master Mix, Applied Biosystems recommends the primer concentrations listed in

“Default Primer Concentrations” below. Detailed discussions follow for the:

• Primer optimization matrix (below)

• TaqMan reagents (below)

• SYBR Green reagents (page 3-31)

Default Primer

Concentrations

Primer

Optimization

Matrix

Taq Ma n

Reagents

The recommended starting primer concentrations listed in the table below are for DNA and cDNA quantitation assays.

Concentrations (nM)

Reagent

Forward Primer Reverse Primer

Ta q Ma n

SYBR

reagents 900 900

Green reagents 50 50

A primer optimization matrix allows you to determine that the minimum primer concentration yields the minimum C

and maximum ΔRn.

A primer optimization matrix can help to compensate for nonspecific primer binding, which can reduce the amount of primer available to bind at its specific site.

For quantitation assays using TaqMan reagents, you can achieve optimal performance by selecting the primer concentrations that provide the lowest C

and

highest ΔRn for a fixed quantity of target template.

Note: Although C

values are the parameter by which quantitative values are

assigned in a real-time quantitation assays, ΔRn values can also be important when you are trying to obtain maximum sensitivity and reproducibility.

The results of a typical TaqMan reagent primer optimization matrix experiment are shown in Figure 3-4 on page 3-31:

• Figure 3-4a shows the amplification plots for all primer concentration combinations in linear view.

• Figure 3-4b shows the same data in log view format.

The combination of 50-nM forward and reverse primer (Plot group C) gives both the lowest ΔRn and highest C

. All other primer combinations that contain a 150-nM

concentration of either the forward or reverse primer (Plot group B) give a reduced ΔRn. All primer combinations that contain at least 300-nM forward and reverse primer (Plot group A) give both the highest ΔRn and the lowest C

; as a result, any of

the plot group A or B provides optimal performance.

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Chapter 3 Quantitation Experiments

a) Linear view

b) Log view

SYBR Green

Reagents

Figure 3-4 Primer optimization experimental results showing amplification plots (linear and log views) of primer combinations Plot group key: A: Combinations that contain at least 300 nM of forward and reverse primer B: Combinations that contain at least 150 nM of forward and reverse primer C: Combinations that contain at least 50 nM of forward and reverse primer

Optimizing primer concentrations is slightly more complex for quantitation assays using SYBR Green reagents. You should perform the same primer optimization matrix as for TaqMan reagents; however, you must include negative controls for SYBR Green reagents. The primer concentrations you select should provide a low C

and high ΔRn when run against the target template, but should not produce

nonspecific product formation with negative controls.

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Chapter 3 Quantitation Experiments

Ta rg e t Amplification

Target Amplification

NC (nonspecific amplification)

(non-specific

amplification)

a) Amplification plot, linear view

b) Melt curve analysis

Melt curves or gel analysis can be extremely useful when you select optimal primer concentrations for quantitation assays using SYBR Green reagents. Figure 3-5 on

page 3-32 shows the results from a primer optimization matrix at primer

concentrations of 900-nM forward and reverse primers:

• Figure 3-5a shows strong amplification of the negative control wells, which indicates that significant nonspecific amplification is occurring.

• Figure 3-5b shows that the melting temperature of the product generated in the absence of template is lower than the melting temperature of the specific product generated with template, indicating that significant nonspecific amplification is occurring.

The results shown in Figure 3-5 are typical of primer-dimer formation. These results indicate that lower primer concentrations may provide more optimal results. Additionally, you can redesign another set of primers to the target of interest.

Figure 3-5 Amplification data using SYBR® Green reagents (a) Amplification plot (linear view) demonstrating suspected nonspecific amplification in negative control (NC) wells (b) Melt curve analysis confirming that product in NC wells has a different melting temperature from the specific product

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Optimize the Probe Concentration

For detection by TaqMan® probes, the recommended probe concentration of 250 nM ensures excellent assay performance. However, depending on the requirements of the assay, a probe optimization experiment can prove useful.

Note: No probe is required for SYBR Green I dye detection.

Chapter 3 Quantitation Experiments

Recommended

Probe

Concentrations

The recommended probe concentrations for DNA and cDNA quantitation assays using TaqMan reagents is 250 nM. Figure 3-6 shows the results of a probe optimization experiment in which the probe concentration is varied from 50 to 250 nM:

• Figure 3-6a shows an increase in ΔRn as the probe concentration is increased.

• Figure 3-6b shows that the C

value changes with sufficient probe

concentrations.

To ensure the best reproducibility, especially when you want to detect low copy numbers of a target, avoid probe limiting concentrations. Run the assay at a probe concentration of 250 nM. Using a 250-nM concentration, you avoid probe limitation and ensure large ΔRn values. Large ΔRn values indicate a robust assay that is performing at high eff iciency, giving high product yield and allowing accurate peak measurement.

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a) Linear view

b) Log view

Chapter 3 Quantitation Experiments

Figure 3-6 Amplification plot (linear and log views) of probe concentration titration from 50 to 250 nM

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For More Information

For information on:

• Using the TaqMan reagents, refer to:

• Using the SYBR Green reagents, refer to:

• Performing quantitation experiments on the StepOne and StepOnePlus systems,

• Performing presence/absence experiments on the StepOne and StepOnePlus

Chapter 3 Quantitation Experiments

– TaqMan –TaqMan –TaqMan

– Power SYBR – SYBR – SYBR

Fast Universal PCR Master Mix (2✕) Protocol

Gene Expression Master Mix Protocol

Universal PCR Master Mix Protocol

Green PCR Master Mix and RT-PCR Protocol

Green PCR Master Mix and RT-PCR Reagents Protocol

Green PCR and RT-PCR Reagents Protocol

refer to: – Applied Biosystems StepOne

™

and StepOnePlus™ Real-Time PCR Systems

Getting Started Guide for Standard Curve Experiments

– Applied Biosystems StepOne

™

and StepOnePlus™ Real-Time PCR Systems Getting Started Guide for Relative Standard Curve and Comparative C Experiments

systems, refer to the Applied Biosystems StepOne

™

and StepOnePlus™ Real-Time PCR Systems Getting Started Guide for Presence/Absence Experiments.

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Chapter 3 Quantitation Experiments

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Genotyping Experiments 4

This chapter covers:

Section 4.1 About Genotyping Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Section 4.2 Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

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Chapter 4 Genotyping Experiments

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Chapter 4 Genotyping Experiments

Section 4.1 About Genotyping Experiments

This section covers:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

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Chapter 4 Genotyping Experiments

Overview

What Is a

Genotyping

Experiment?

A genotyping experiment is an endpoint experiment used to determine the genotype of unknown samples. With this experiment type, you can differentiate a single nucleotide polymorphism (SNP).

A genotyping experiment determines if unknown samples are:

• Homozygotes (samples having only allele 1)

• Homozygotes (samples having only allele 2)

• Heterozygotes (samples having both allele 1 and allele 2)

Components

PCR reactions for genotyping experiments include the following components:

• Sample – The sample in which the genotype of the target is unknown.

• (Optional) Replicates – Identical reactions containing identical components and volumes.

• Negative Controls – Samples that contain water or buffer instead of template; also known as no template controls (NTCs). Negative controls should not amplify.

• (Optional) Positive controls – Samples that contain known genotypes (homozygotes for allele 1, homozygotes for allele 2, and heterozygotes for alleles 1 and 2).

Instruments

How Genotyping

Experiments

Work

Genotyping experiments require two steps: thermal cycling (PCR amplification) followed by endpoint detection of the resulting fluorescent signals. You can perform the thermal cycling step (PCR amplification) on the Applied Biosystems StepOne and StepOnePlus

If you use the StepOne

™

Real-Time PCR Systems or on a standalone thermal cycler.

™

and StepOnePlus™ systems:

™

• You can analyze the PCR, which is helpful for troubleshooting.

• Perform the endpoint plate read separately.

In genotyping experiments, the PCR includes a specific fluorescent-dye-labeled probe for each allele. The probes contain different fluorescent reporter dyes to differentiate each allele.

You can use TaqMan StepOnePlus systems. Each TaqMan

minor groove binder (MGB) probes on the StepOne and

MGB probe contains:

• A reporter dye at the 5′ end of each probe

–VIC –FAM

dye is linked to the 5′ end of the Allele 1 probe

™

dye is linked to the 5′ end of the Allele 2 probe

• A minor groove binder (MGB) This modification increases the melting temperature (Tm) of probes without

increasing probe length (Afonina et al., 1997; Kutyavin et al., 1997), thereby allowing the design of shorter probes. Consequently, the TaqMan MGB probes exhibit greater differences in Tm values between matched and mismatched probes; greater differences in Tm values provide accurate genotyping.

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Chapter 4 Genotyping Experiments

• A nonfluorescent quencher (NFQ) at the 3′ end of the probe

Because the quencher does not fluoresce, real-time PCR systems can measure reporter dye contributions more accurately.

During PCR, each probe anneals specifically to its complementary sequence between the forward and reverse primer sites. AmpliTaq Gold

DNA polymerase can cleave only probes that hybridize to the allele sequence (match). Cleavage separates the reporter dye from the quencher dye, increasing fluorescence of the reporter dye. Thus, the fluorescence signals generated during PCR amplification indicate the alleles that are present in the sample.

Mismatches Between Probe and Allele Sequences

Mismatches between a probe and allele (Figure 4-1) reduce the efficiency of probe hybridization. Furthermore, AmpliTaq Gold DNA polymerase is likely to displace the mismatched probe rather than to cleave it to release reporter dye.

Allele

Match

Mismatch

Legend

VIC® dye

FAM ™ dye

Quencher

AmpliTaq

Gold

DNA

Polymerase

GR1556

Workflow

Figure 4-1 Results from matches and mismatches between allele and probe sequences in genotyping experiments

Tabl e 4- 1 summarizes the possible results of the genotyping experiment example

shown above.

Table 4-1 Genotyping experiment results

A substantial increase in… Indicates…

VIC® dye fluorescence only homozygosity for Allele 1.

™

dye fluorescence only homozygosity for Allele 2.

FAM

both fluorescent signals heterozygosity.

Before performing genotyping experiments on the StepOne and StepOnePlus systems, prepare for the experiment as follows:

1. Select the assay type (below).

2. Review the design guidelines for the assay type you selected (Section 4.2 on

page 4-9).

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Chapter 4 Genotyping Experiments

Select the Assay Type

When you design your experiments with the StepOne software, you can select the following assay types for genotyping experiments:

• Pre-Designed/Validated (below)

•Custom (page 4-7)

This section lists the products available for each assay type.

Note: The assays are specific to the target of interest. The master mixes contain the

remaining components needed for the PCR reaction.

Pre-Designed/

Validated Assays

Product Attributes

Ta q Ma n Genotyping Assays

Ta q Ma n Metabolism Genotyping Assays

Pre-Developed TaqMan Assay Reagents for Allelic Discrimination

Ta q Ma n Master Mix

Ta q Ma n PCR Master Mix (with or without AmpErase

SNP

Drug

Genotyping

2✕ Universal

UNG)

• Predesigned assays for high-density, genome-wide marker coverage.

• For screening, association, candidate region, candidate gene, or fine-mapping studies.

• Convenient single-tube format.

• Detect polymorphisms in 220 genes that code for various drug metabolism enzymes and drug transporters.

• For studying single-nucleotide polymorphisms (SNPs), insertions/deletions (in/dels), and multinucleotide polymorphisms (MNPs).

• Genotype purified DNA samples for specific mutations; most assays discriminate between two alleles of singlenucleotide polymorphisms (SNPs).

• Minor groove binder (MGB) added for better genotyping.

• Allele 1 and 2 control DNA included to allow each homozygote signal to be generated on each run.

• Closed-tube system requires no post-PCR manipulation or gels.

Optimized for endpoint fluorescence detection in SNP genotyping applications:

• Distinct clusters and high call rates for unambiguous allelic discrimination.

• Excellent pre- and post-PCR stability for high-throughput setup and analysis.

• Validated with TaqMan

• Simplifies assay implementation by using one reagent for all assays.

• Provides optimal performance for TaqMan use cDNA or DNA as a template.

• Contains components that ensure excellent assay performance.

• Simplifies assay implementation by using one reagent for all assays.

SNP Genotyping Assays.

assays that

Note: Genotyping experiments are not supported for Fast or SYBR

Green master

mixes and protocols.

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Chapter 4 Genotyping Experiments

For guidelines on designing your experiments with Pre-Designed/Validated assays, see page 4-10.

Custom Assays

Product Attributes

Custom TaqMan

SNP

Genotyping Assays

Ta q Ma n

Genotyping

Master Mix

Ta q Ma n

2✕ Universal PCR Master Mix (with or without AmpErase

UNG)

• Any possible single-nucleotide polymorphism (SNP) in any organism.

• Detect insertions/deletions (in/dels) of up to six bases.

• Detect multiple nucleotide polymorphisms (MNPs) of up to six bases.

• Convenient single-tube format.

Optimized for endpoint fluorescence detection in SNP genotyping applications:

• Distinct clusters and high call rates for unambiguous allelic discrimination.

• Excellent pre- and post-PCR stability for high-throughput setup and analysis.

• Validated with TaqMan

SNP Genotyping Assays.

• Simplifies assay implementation by using one reagent for all assays.

• Provides optimal performance for TaqMan

assays that

use cDNA or DNA as a template.

• Contains components that ensure excellent assay performance.

• Simplifies assay implementation by using one reagent for all assays.

Note: Genotyping experiments are not supported for Fast or SYBR

Green master

mixes and protocols.

For guidelines on designing your experiments with Custom assays, see page 4-14.

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Section 4.2 Design Guidelines

This section covers:

Pre-Designed/Validated Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

Taq Ma n TaqMa n Pre-Developed TaqMan

Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Custom TaqMan

Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16

Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16

SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

Drug Metabolism Genotyping Assays . . . . . . . . . . . . . . . . . . . . 4-11

Assay Reagents for Allelic Discrimination . . . . 4-12

SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . 4-14

Chapter 4 Genotyping Experiments

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Chapter 4 Genotyping Experiments

Pre-Designed/Validated Assays

Workflow

If you are designing a genotyping experiment using Applied Biosystems PreDesigned/Validated assays, Applied Biosystems recommends that you follow the workflow below:

1. Select the assay:

•TaqMan

SNP Genotyping Assays (below).

Drug Metabolism Genotyping Assays (page 4-11).

Pre-Developed Assays Reagents for Allelic Discrimination

(page 4-12).

2. Select the master mix (page 4-13).

3. Design the experiment using the StepOne

TaqM an® SNP Genotyping Assays

Product

Description

Taq Ma n® SNP Genotyping Assays are a comprehensive collection of primer and probe sets for genotyping single nucleotide polymorphisms (SNPs) for human studies.

The assays:

•Use TaqMan

reagents to amplify and detect specific SNP alleles in purif ied

genomic DNA samples.

• Are designed using Applied Biosystems bioinformatics pipeline and software, as well as genomic information from Celera Genomics and public databases.

• Are designed and optimized to work with an Applied Biosystems TaqMan® master mix, using universal thermal cycling conditions.

™

software (page 4-14).

Product

Requirements

All TaqMan SNP Genotyping Assays require:

• Three components: – 1 to 20 ng of purified genomic DNA sample. –20✕, 40✕, or 80✕ SNP Genotyping Assay Mix (specific for each

polymorphism). Each assay consists of sequence-specific forward and reverse primers to amplify the SNP of interest and two TaqMan MGB probes: One probe labeled with VIC probe labeled with FAM

–TaqMan

Genotyping Master Mix or TaqMan® 2✕ Universal PCR Master

™

dye detects the Allele 2 sequence.

Mix (with or without AmpErase

dye detects the Allele 1 sequence; one

UNG).

• PCR amplification and an endpoint read to obtain results.

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Chapter 4 Genotyping Experiments

Available Assays

For More

Information

TaqMan SNP Genotyping Assays are available as TaqMan® Pre-Designed SNP Genotyping Assays:

• Over 4.5 million predesigned genome-wide assays, including 3.5 million human HapMap SNPs, 70,000 human cSNPs, 160,000 human validated, and 10,000 mouse assays.

• Available in small, medium, and large scale.

• Made to Order (that is, manufactured at the time of order).

• For information on the latest available products and specific product uses, refer to the Applied Biosystems Web sites:

http://www.allsnps.com/

and/or

http://www.appliedbiosystems.com/

a. On the Home page, under TaqMan

Products, select TaqM an® SNP

Genotyping Assays.

b. On the SNP Genotyping Assays page, under Pre-Designed/Validated

Assays, select Ta qM an

SNP Genotyping Assays.

• For information on preparing PCR reactions using the TaqMan SNP Genotyping Assays, refer to the TaqMan

SNP Genotyping Assays Protocol.

TaqM an® Drug Metabolism Genotyping Assays

Product

Description

Product

Requirements

Taq Ma n® Drug Metabolism Genotyping Assays are a comprehensive collection of Inventoried primer and probe sets for genotyping SNPs, insertions and deletions (indels), and multiple nucleotide polymorphisms (MNPs) in drug metabolism related genes.

The assays:

• Use TaqMan reagents to amplify and detect specific polymorphisms in purified genomic DNA samples.

• Are designed using Applied Biosystems bioinformatics pipeline and software, as well as genomic information from public SNP databases and public genome assemblies.

• Are designed and optimized to work with Applied Biosystems TaqMan mixes.

All TaqMan Drug Metabolism Genotyping Assays require:

• Three components: – 3 to 30 ng of purified genomic DNA sample. –20✕ Drug Metabolism Genotyping Assay Mix (specific for each

polymorphism). Each assay consists of sequence-specific forward and reverse primers to amplify the polymorphic sequence of interest and two TaqMan MGB probes: One probe labeled with VIC dye detects the Allele 1 sequence; one probe labeled with FAM dye detects the Allele 2 sequence.

–TaqMan

Mix (with or without AmpErase

Genotyping Master Mix or TaqMan® 2✕ Universal PCR Master

UNG).

master

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• PCR amplification and an endpoint read to obtain results.

For More

Information

• For information on the latest available products and specific product uses, refer to the Applied Biosystems Web sites:

http://www.allsnps.com/

and/or

http://www.appliedbiosystems.com/

a. On the Home page, under TaqMan

Products, select Taq Ma n® SNP

Genotyping Assays.

b. On the SNP Genotyping Assays page, under Pre-Designed/Validated

Assays, select Ta qM an

Drug Metabolism Genotyping Assays.

• For information on preparing PCR reactions using the TaqMan SNP Genotyping Assays, refer to the TaqMan

Drug Metabolism Genotyping Assays Protocol.

Pre-Developed TaqMan® Assay Reagents for Allelic Discrimination

Product

Description

Pre-Developed TaqMan® Assay Reagents for Allelic Discrimination (TaqMan® PDARs for AD) are Inventoried assays optimized for the discrimination of specific alleles.

The assays:

• Use TaqMan reagents to amplify and detect specific polymorphisms in purified genomic DNA samples.

• Are designed and optimized to work with an Applied Biosystems TaqMan master mix, using universal thermal cycling conditions.

Product

Requirements

For More

Information

TaqMan PDARs for AD require three components:

• 2 to 20 ng of purified genomic DNA sample.

•10✕ Allelic Discrimination Assay Mix (specific for each polymorphism). Each assay consists of sequence-specific forward and reverse primers to amplify the polymorphic sequence of interest and two TaqMan MGB probes: One probe labeled with VIC dye detects the Allele 1 sequence; one probe labeled with FAM dye detects the Allele 2 sequence.

•TaqMan (with or without AmpErase

Note: Allele 1 and 2 control DNA is included with each assay to allow each

Genotyping Master Mix or TaqMan® 2✕ Universal PCR Master Mix

UNG).

homozygote signal to be generated on each run.

• For information on the latest available products and specific product uses, refer to the Applied Biosystems Web sites:

http://www.allsnps.com/

and/or

http://www.appliedbiosystems.com/

a. On the Home page, under TaqMan

Products, select Taq Ma n® SNP

Genotyping Assays.

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• For information on preparing PCR reactions using the TaqMan PDARs for

Select the Master Mix

Available Master

Mixes

Applied Biosystems Pre-Designed/Validated assays for genotyping experiments are designed to work with the following master mixes:

• TaqMan PDARs for AD contain TaqMan

• TaqMan SNP Genotyping Assays and Custom TaqMan SNP Genotyping

b. On the SNP Genotyping Assays page, under Pre-Designed/Validated

Assays, select Ta qM an

Discrimination (TaqMan

Allelic Discrimination, refer to the Pre-Developed TaqMan

Pre-Developed Assay Reagents for Allelic

PDARs for AD).

Assay Reagents

Allelic Discrimination Protocol.

2✕ Universal PCR Master Mix

(with AmpErase

UNG).

Assays can be used with:

Master Mix Part Number

Ta qM an 400 reactions

Ta qM an 2000 reactions

Ta qM an

10-Pack, TaqMan

Ta qM an UNG, 200 Reactions

Ta qM an UNG, 2000 Reactions

10-Pack, TaqMan AmpErase

Genotyping Master Mix, 1-Pack (1 × 10 mL),

Genotyping Master Mix, 1 Bulk Pack (1 × 50 mL),

2✕ Universal PCR Master Mix, 200 Reactions 4304437

2✕ Universal PCR Master Mix, 2000 Reactions 4326708

2✕ Universal PCR Master Mix 4305719

2✕ Universal PCR Master Mix, No AmpErase®

2✕ Universal PCR Master Mix, No

UNG

4371355

4371357

4324018

4326614

4324020

For More

Information

Note: Genotyping experiments are not supported for Fast or SYBR

mixes and protocols.

For information on using the TaqMan master mixes, refer to the:

• TaqMan

Genotyping Master Mix Protocol

Universal PCR Master Mix Protocol

Green master

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Design the Experiment

Use the StepOne

Software

For Applied Biosystems Pre-Designed/Validated assays, use the StepOne software to design your genotyping experiments. The StepOne software automatically calculates volumes for the:

Note: To select a SNP assay in the StepOne software, go to the SNP Assays screen in

the Design Wizard or to the Plate Setup screen in Advanced Setup. In the SNP Assays screen or Plate Setup screen, you can select an assay from the library or create a new assay.

For More

Information

For information on designing and performing genotyping experiments on the StepOne and StepOnePlus systems, refer to Applied Biosystems StepOne

StepOnePlus Experiments.

Custom Assays

Workflow

If you are designing a genotyping experiment using Applied Biosystems Custom assays, Applied Biosystems recommends that you follow the workflow below:

1. Order the assay: Custom TaqMan

• Reaction mix components

• Sample dilutions

™

Real-Time PCR Systems Getting Started Guide for Genotyping

SNP Genotyping Assays (below).

™

and

2. Select the master mix (page 4-16).

3. Design the experiment using the StepOne software (page 4-16).

Custom TaqMan® SNP Genotyping Assays

Product

Description

Custom TaqMan® SNP Genotyping Assays are TaqMan probe and primer sets that are designed, synthesized, and formulated by the Custom TaqMan service based on sequence information that you submit. Custom TaqMan SNP Genotyping Assays allow you to:

Action Example

Perform genotyping studies with any possible single-nucleotide polymorphism (SNP) in any organism

Detect insertions/deletions (in/dels) of up to six bases for genotyping studies

Detect multiple nucleotide polymorphisms (MNPs) of up to six bases for genotyping studies

Genomic Assays

AGTTCATCCATGGTCA --> AGTTCATACATGGTCA

Annotated as: AGTTCAT[C/A]CATGGTCA

AGTTCATCCATGGTCA --> AGTTCATGGTCA

Annotated as: AGTTCAT[CCAT/*]GGTCA

AGTTCATCCGGTCA --> AGTTCATATGGTCA

Annotated as: AGTTCAT[CC/AT]GGTCA

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The assays:

• Use TaqMan reagents to amplify and detect specific polymorphisms in purified genomic DNA (gDNA).

• Are developed using proprietary assay-design software.

• Are designed and optimized to work with an Applied Biosystems TaqMan master mix, using universal thermal cycling conditions.

Product

Requirements

For More

Information

All Custom TaqMan SNP Genotyping Assays require:

• A submission file that includes your target SNP sequence. You create the submission file using free File Builder software, then submit the file to the Custom TaqMan

Genomic Assays service.

• Three components: – 1 to 20 ng of purified gDNA sample per well. –40✕ SNP Genotyping Assay or 80✕ SNP Genotyping Assay (specific for

each polymorphism). Each assay consists of sequence-specific forward and reverse primers to amplify the SNP of interest and two TaqMan MGB probes: One probe labeled with VIC dye detects the Allele 1 sequence; one probe labeled with FAM dye detects the Allele 2 sequence.

–TaqMan

(with or without AmpErase

Genotyping Master Mix or TaqMan® Universal PCR Master Mix

UNG).

• PCR amplification and an endpoint read to obtain results.

• For information on the latest available products and specific product uses, refer to the Applied Biosystems Web sites:

http://www.allsnps.com/

and/or

http://www.appliedbiosystems.com/

a. On the Home page, under TaqMan

Products, select TaqM an® SNP

Genotyping Assays.

b. On the SNP Genotyping Assays page, under Custom Assays, select

Custom TaqMan

SNP Genotyping Assays.

• For information on ordering Custom TaqMan SNP Genotyping Assays, refer to the Custom TaqMan

Genomic Assays Protocol: Submission Guidelines.

• For information on preparing PCR reactions using the Custom TaqMan SNP Genotyping Assays, refer to the Custom TaqMan

SNP Genotyping Assays

Protocol.

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Select the Master Mix

Available Master

Mixes

Applied Biosystems Made to Order assays for genotyping experiments are designed to work with the following master mixes:

Master Mix Part Number

Ta q Ma n® Genotyping Master Mix, 1-Pack (1 × 10 mL), 400 reactions 4371355

Ta q Ma n 2000 reactions

Ta q Ma n

10-Pack, TaqMan

Ta q Ma n Reactions

10-Pack, TaqMan UNG

Genotyping Master Mix, 1 Bulk Pack (1 × 50 mL),

2✕ Universal PCR Master Mix, 200 Reactions 4304437

2✕ Universal PCR Master Mix, 2000 Reactions 4326708

2✕ Universal PCR Master Mix 4305719

2✕ Universal PCR Master Mix, No AmpErase® UNG, 200

2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000

2✕ Universal PCR Master Mix, No AmpErase®

Note: Genotyping experiments are not supported for Fast or SYBR

4371357

4324018

4326614

4324020

Green master

mixes and protocols.

For More

Information

For information on using the TaqMan master mixes, refer to the:

• TaqMan

Design the Experiment

Use the StepOne

Software

For More

Information

For Applied Biosystems Custom assays, use the StepOne software to design your genotyping experiments. The StepOne software automatically calculates volumes for the:

• Reaction mix components

• Sample dilutions

Note: To select a SNP assay in the StepOne software, go to the SNP Assays screen

in the Design Wizard or to the Plate Setup screen in Advanced Setup. In the SNP Assays screen or Plate Setup screen, you can select an assay from the library or create a new assay.

For information on designing and performing genotyping experiments on the StepOne and StepOnePlus systems, refer to Applied Biosystems StepOne

StepOnePlus Experiments.

Genotyping Master Mix Protocol

Universal PCR Master Mix Protocol

™

Real-Time PCR Systems Getting Started Guide for Genotyping

and

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Presence/Absence Experiments 5

This chapter covers:

Section 5.1 About Presence/Absence Experiments. . . . . . . . . . . . . . . . . . . . . . . . . 5-3

Section 5.2 Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9

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Chapter 5 Presence/Absence Experiments

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Chapter 5 Presence/Absence Experiments

Section 5.1 About Presence/Absence Experiments

This section covers:

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4

Select the Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6

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Chapter 5 Presence/Absence Experiments

Overview

What Is a

Presence/

Absence

Experiment?

A presence/absence experiment is an endpoint experiment that indicates the presence or absence of a specific nucleic acid sequence (target) in a sample. The actual quantity of target is not determined.

Presence/absence experiments are commonly used to detect the presence or absence of a pathogen, such as a viral or bacterial pathogen. For example, a presence/absence experiment might be used to determine if Salmonella bacteria are present in hamburger meat. The results will show if Salmonella bacteria are present or are not present; the quantity of bacteria is not determined.

Components

PCR reactions for presence/absence experiments include the following components:

• Sample – The sample in which the presence of a target is unknown.

• Replicates – Identical reactions containing identical components and volumes.

• Internal positive control (IPC) – A short synthetic DNA template that is added to PCR reactions. You can use the IPC to distinguish between true negative results and reactions affected by PCR inhibitors, incorrect assay setup, or a reagent or instrument failure.

Note: Presence/absence experiments can be performed without an IPC;

however, the IPC ensures that a failed PCR is not mistaken for a negative test result.

• Negative Controls – Wells that contain water or buffer instead of sample template. No amplification of the target should occur in negative control wells.

In the StepOne software, you can set up the PCR reactions for the presence/absence experiments three different ways:

Setup Well Types

IPC setup There are three types of wells:

• Unknown-IPC wells – Wells contain sample template and IPC template; the presence of the target is not known.

• Negative control-IPC wells – Wells contain IPC template and water or buffer instead of sample template in the PCR reaction. Only the IPC template should amplify in negative control-IPC wells because the reaction contains no sample template. Also called IPC+.

• Negative control-blocked IPC wells – Wells contain IPC blocking agent instead of sample template in the PCR reaction. No amplification should occur in negative control-blocked IPC wells because the reaction contains no sample template and amplification of the IPC is blocked. Also called no amplification control (NAC).

No IPC, singleplex setup

There are two types of wells:

• Unknown wells – Wells contain sample template; the presence of the target is not known.

• Negative controls –Wells contain water or buffer instead of sample template.

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Chapter 5 Presence/Absence Experiments

Setup Well Types

Endpoint

Detection and

Post-PCR Plate

Read

How Presence/

Absence

Experiments

Work

No IPC, multiplex setup

There are two types of wells:

• Unknown-Unknown wells – Wells contain sample template; the presence of the target is not known.

• Negative control-Negative control wells – Wells contain water or buffer instead of sample template.

Presence/absence experiments are endpoint experiments in which fluorescence data are collected after the PCR is complete.

To aid in troubleshooting presence/absence experiments, you can use the Applied

™

Biosystems StepOne time PCR. If you use the StepOne

and StepOnePlus™ Real-Time PCR Systems to perform real-

™

and StepOnePlus™ systems for PCR

amplification, perform the pre-PCR read and post-PCR read runs separately.

During the PCR, the fluorogenic probes anneal specifically to the complementary target between the forward and reverse primer sites on the template DNA. Then during extension, AmpliTaq Gold

DNA polymerase cleaves the hybridized probes in each sample containing the target. The cleavage of each matched probe separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter.

™

After PCR cycling, the StepOne

and StepOnePlus™ instruments read the fluorescence generated during the PCR amplification. The fluorescent signals are used to determine the presence or absence of the target in each sample. Reporter signals are normalized to the emission of a passive reference, as follows:

Incorporating an IPC

= Emission Intensity of Target Template Sequence

n (TT)

Emission Intensity of Passive Reference

= Emission Intensity of Internal Positive Control

n (IPC)

Emission Intensity of Passive Reference

An IPC is a second TaqMan® probe and primer set added to the reaction plate to detect a low-copy, constitutive nucleic acid. The IPC and the target are amplified simultaneously in the same reaction well. If a well does not exhibit amplification, the StepOne

™

software uses the positive signal from the IPC to confirm that the well failed to amplify because of a lack of target template, rather than because of a pipetting error or inhibition.

Note: Presence/absence experiments can be performed without an IPC; however, the

IPC ensures that a failed PCR is not mistaken for a negative test result.

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Chapter 5 Presence/Absence Experiments

Workflow

Before performing presence/absence experiments on the StepOne and StepOnePlus systems, prepare for the experiment as follows:

1. Select the assay type (below).

2. Review the design guidelines for the assay type you selected (Section 5.2 on

page 5-9).

Select the Assay Type

When you design your experiments with the StepOne software, you can select the following assay types for presence/absence experiments:

• Inventoried/Made to Order (page 5-7)

•Custom (page 5-8)

This section lists the products available for each assay type.

Note: The assays are specific to the target of interest. The master mixes contain the

remaining components needed for the PCR reaction.

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Chapter 5 Presence/Absence Experiments

Inventoried/Made

to Order Assays

Product Attributes

Ta q Ma n

Gene

Expression Assays

Ta q Ma n

Endogenous

Control Assays

Note: FAM

™

dyelabeled TaqMan® Endogenous Control Assays are included as Inventoried TaqMan Gene Expression Assays.

Custom TaqMan

Gene

Expression Assays

Ta q Ma n

Gene

Expression Master Mix

Ta q Ma n

2✕ Universal PCR Master Mix (with or without AmpErase

UNG)

• Predesigned, gene-specific primer and probe sets for human, mouse, rat, Arabidopsis, Drosophila, C. elegans, C. familiares (dog), and M. mulatta (Rhesus) genes.

™

•Probe is a FAM

dye-labeled MGB probe.

• Provided in a convenient, single, 205 tube.

• Available as Inventoried or Made to Order assays.

• Optimized, preformulated, ready-to-use endogenous control assays.

• Cost-effective gene expression quantitation for human, mouse, rat, Arabidopsis, Drosophila, and any eukaryotic species.

™

•Choice of FAM

dye or VIC® dye labels (primer-limited).

• Any species or organism.

• Target of your choice.

•Probe is a FAM™ dye-labeled MGB probe.

• Provided in a convenient, single, 205 tube.

Tailored for precise quantitation by real-time PCR for routine and challenging experiments:

• Sensitive detection down to 1 copy of target.

• Multiplex PCR for co-amplifying two targets in a single reaction.

• Specificity for differentiation between gene family members.

• Validated with TaqMan

Gene Expression Assays.

• Simplifies assay implementation by using one reagent for all assays.

• Provides optimal performance for TaqMan

assays that

use cDNA or DNA as a template.

• Contains components that ensure excellent assay performance.

• Simplifies assay implementation by using one reagent for all assays.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

Note: Presence/absence experiments are not supported for Fast or SYBR

Green

master mixes and protocols.

For guidelines on designing your experiments with Inventoried/Made to Order assays, see page 5-10.

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Chapter 5 Presence/Absence Experiments

Custom Assays

Product Attributes

Custom TaqMan

Probes

and Primers

Primer Express

Software

Ta q Ma n

Gene

Expression Master Mix

Ta q Ma n

2✕ Universal PCR Master Mix (with or without AmpErase

UNG)

• Any species or organism.

• Choice of dye labels, quenchers, and synthesis scales.

• For use with the Primer Express® Software and Applied Biosystems Assay Design Guidelines.

Software that designs primers and probes for real-time PCR.

Tailored for precise quantitation by real-time PCR for routine and challenging experiments:

• Sensitive detection down to 1 copy of target.

• Multiplex PCR for co-amplifying two targets in a single reaction.

• Specificity for differentiation between gene family members.

• Validated with TaqMan

Gene Expression Assays.

• Simplifies assay implementation by using one reagent for all assays.

• Provides optimal performance for TaqMan

assays that

use cDNA or DNA as a template.

• Contains components that ensure excellent assay performance.

• Simplifies assay implementation by using one reagent for all assays.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

Note: Presence/absence experiments are not supported for Fast or SYBR

Green

master mixes and protocols.

For guidelines on designing your experiments with Custom assays, see page 5-16.

5-8 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide

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Section 5.2 Design Guidelines

This section covers:

Inventoried/Made to Order Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10

Taq Ma n Custom TaqMan Taq Ma n

Select the Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15

Design the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16

Custom Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-16

Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10

Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . 5-12

Exogenous Internal Positive Control Reagents. . . . . . . . . . . . . . 5-13

Chapter 5 Presence/Absence Experiments

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Chapter 5 Presence/Absence Experiments

Inventoried/Made to Order Assays

Workflow

If you select the Inventoried/Made to Order assay type in the StepOne™ software, Applied Biosystems recommends that you follow the workflow below:

1. Select the assay:

•TaqMan

Gene Expression Assays (below).

• Custom TaqMan

2. Use the TaqMan

Note: Presence/absence experiments can be performed without an IPC;

Exogenous Internal Positive Control Reagents (page 5-13).

however, the IPC ensures that a failed PCR is not mistaken for a negative test result.

3. Select the master mix (page 5-15).

4. Design the experiment using the StepOne software (page 5-16).

TaqM an® Gene Expression Assays

Product

Description

Taq Ma n® Gene Expression Assays are a comprehensive collection of Inventoried and Made to Order probe and primer sets that you can use to perform presence/absence experiments on human, mouse, rat, Arabidopsis, Drosophila, C. elegans, C. familiares (dog), and M. mulatta (Rhesus) genes.

The assays:

•Use TaqMan

reagents to amplify and detect the target in cDNA samples.

• Are designed and optimized to work with an Applied Biosystems TaqMan master mix, using universal thermal cycling conditions.

• When possible, amplify target cDNA without amplifying genomic DNA (m suffix in assay ID) by designing probes that cross exon-exon junctions.

Gene Expression Assays (page 5-12).

Product

Requirements

All TaqMan Gene Expression Assays require:

• Three components: – 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells

in a study having the same amount of cDNA.

–20✕ Gene Expression Assay Mix (specific for each target). Each assay mix

consists of two unlabeled PCR primers and a FAM

™

dye-labeled TaqMan® MGB (minor groove binder) probe in a preformulated 20✕ mix. 1✕ final concentrations are 250 nM for the probe and 900 nM for each primer.

–TaqMan

Gene Expression Master Mix or TaqMan Universal PCR Master

Mix (with or without AmpErase UNG).

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Chapter 5 Presence/Absence Experiments

Available Assays

TaqMan Gene Expression Assays are available for human, mouse, rat, Arabidopsis, Drosophila, C. elegans, C. familiares (dog), and M. mulatta (Rhesus) genes. The part

numbers are:

• PN 4331182 for Inventoried assays

• PN 4351372 for Made to Order assays

The prefix of the assay name indicates the species for which the assay was designed:

Hs for Homo sapiens (human), Mm for Mus musculus (mouse), Rn for Rattus norvegicus (rat), At for Arabidopsis thaliana, Dm for Drosophila melanogaster,

Ce for C. elegans, Cf for C. familiares (dog), and Rh for M. mulatta (Rhesus).

The suffix of the assay name indicates the assay placement, as described in the table below.

Suffix Description

_m The assay’s probe spans an exon junction; the assay does not detect

genomic DNA.

_s The assay’s primers and probes are designed within a single exon; the assay

does detect genomic DNA.

_g The assay may detect genomic DNA; the assay’s primers and probes may be

within a single exon.

_mH The assay was designed to a transcript belonging to a gene family with high

_sH

_gH

_u The assay’s amplicon spans an exon junction and the probe sits completely in

one of the spanned exons.

and 15 CT difference

Taq Ma n® Endogenous Control Assays

TaqMa n® Endogenous Control Assays are available as:

• Inventoried TaqMan Gene Expression Assays (PN 4331182) – Each assay

™

contains a FAM

dye-labeled TaqMan® MGB probe in a single, preformulated

20✕ tube.

• Individual control assays for all human, mouse, and rat species (various part numbers) – Each assay contains either a FAM probe, a VIC

dye-labeled TaqMan® MGB probe, or a TAMRA™ dye-labeled

™

dye-labeled TaqMan® MGB

probe. TaqMan Endogenous Controls with VIC dye labels are primer-limited.

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

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Chapter 5 Presence/Absence Experiments

For More

Information

• For information on the latest available products and specific product uses, refer to the Applied Biosystems Web site:

http://www.appliedbiosystems.com/

a. On the Home page, under TaqMan

Products, select Taq Ma n® Gene

Expression Assays.

b. On the Gene Expression Assays & Arrays page:

• Under Individual Assays, select Taq Man

Gene Expression Assays.

This option links to all TaqMan Gene Expression Assays, including TaqMan Endogenous Control Assays that contain FAM dye-labeled probes.

• Under Individual Control Assays, select Ta qM a n

Endogenous

Control Assays. This option links to the individual TaqMan

Endogenous Control Assays (that contain FAM dye-labeled TaqMan MGB probes, VIC dye-labeled TaqMan MGB probes, or TAMRA dye-labeled probes).

• For information on Custom TaqMan Endogenous Control Assays, refer to the

Using TaqMan

Endogenous Control Assays to Select an Endogenous Control

for Experimental Studies Application Note.

• For information on preparing PCR reactions using the TaqMan Gene Expression Assays, refer to the TaqMan

Gene Expression Assays Protocol.

Custom TaqMan® Gene Expression Assays

Product

Description

Product

Requirements

Custom TaqMan® Gene Expression Assays are TaqMan probe and primer sets that are designed, synthesized, and formulated by the Custom TaqMan service based on sequence information that you submit. Custom TaqMan Gene Expression Assays allow you to perform presence/absence experiments on any gene or splice variant in any organism.

The assays:

•Use TaqMan

reagents to amplify and detect the target in cDNA samples.

• Are developed using proprietary assay-design software.

• Are designed and optimized to work with an Applied Biosystems TaqMan master mix, using universal thermal cycling conditions.

All Custom TaqMan Gene Expression Assays require:

• A submission file that includes your target sequence. You create the submission file using free File Builder software, then submit the file to the Custom

Taq Ma n

Genomic Assays service.

• Three components: – 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells

in a study having the same amount of cDNA.

–20✕ Gene Expression Assay or 60✕ Gene Expression Assay (specific for

Genomic Assays

™

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Chapter 5 Presence/Absence Experiments

–TaqMan® Gene Expression Master Mix or TaqMan Universal PCR Master

Mix (with or without AmpErase UNG).

For More

Information

• For information on the latest available products and specific product uses, refer to the Applied Biosystems Web site:

http://www.appliedbiosystems.com/

a. On the Home page, under TaqMan

Products, select TaqM an® Gene

Expression Assays.

b. On the Gene Expression Assays & Arrays page, under Individual Assays,

select Custom TaqMan

Gene Expression Assays.

• For information on ordering Custom TaqMan Gene Expression Assays, refer to the Custom TaqMan

Genomic Assays Protocol: Submission Guidelines.

• For information on preparing PCR reactions using the Custom TaqMan Gene Expression Assays, refer to the Custom TaqMan

Protocol.

TaqM an® Exogenous Internal Positive Control Reagents

Product

Description

The Applied Biosystems TaqMan® Exogenous Internal Positive Control Reagents contain:

• An internal positive control (IPC) with predesigned primers and probe

• IPC DNA template

• IPC blocking control

Gene Expression Assays

Product

Requirements

The reagents are designed to:

• Distinguish types of negative results: – A negative call for the target and positive call for the IPC indicates that no

target is present.

– A negative call for the target and negative call for the IPC suggests PCR

inhibition.

• Avoid amplification of endogenous controls.

• Permit co-amplification of the IPC and the target without compromising amplification of the target.

• Detect the IPC using a VIC

• Detect the target using a FAM

• Work with the TaqMan AmpErase

UNG), using universal thermal cycling conditions.

dye-labeled probe.

™

dye-labeled probe.

2✕ Universal PCR Master Mix (with or without

The TaqMan Exogenous Internal Positive Control Reagents kits require the following components:

•DNA sample

• TaqMan assay for your target of interest

•TaqMan 2✕ Universal PCR Master Mix (with or without AmpErase UNG)

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Chapter 5 Presence/Absence Experiments

Available Kits

The following TaqMan Exogenous Internal Positive Control Reagents kits are available from Applied Biosystems:

Kits Part Number

Ta q Ma n® Exogenous Internal Positive Control Reagents with Ta q Ma n

Ta q Ma n

Note: If you are using this kit, you will need to purchase one of these

Ta q Ma n

•TaqMan

2✕ Universal PCR Master Mix (with VIC® dye)

Exogenous Internal Positive Control Reagents

reagents separately:

2✕ Universal PCR Master Mix (PN 4304437)

PCR Core Reagents Kit (PN N808-0228)

4308320

4308323

IMPORTANT! The kits listed above contain TAMRA™ dye-labeled probes; Applied

Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system. The kits may be used with the StepOnePlus system; TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

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Chapter 5 Presence/Absence Experiments

For More

Information

For information on preparing PCR reactions using the TaqMan Exogenous Internal Positive Control Reagents, refer to the TaqMan Reagents Protocol.

Select the Master Mix

Available Master

Mixes

Applied Biosystems Inventoried/Made to Order assays for presence/absence experiments are designed to work with the following master mixes:

Ta q Ma n® Gene Expression Master Mix, 1-Pack (1 × 5 mL), 200 reactions

Ta q Ma n 2000 reactions

Ta q Ma n

10-Pack, TaqMan

Ta q Ma n Reactions

10-Pack, TaqMan UNG

Ta q Ma n

Exogenous Internal Positive Control

Master Mix Part Number

4369016

Gene Expression Master Mix, 10-Pack (10 × 5 mL),

2✕ Universal PCR Master Mix, 200 Reactions 4304437

2✕ Universal PCR Master Mix, 2000 Reactions 4326708

2✕ Universal PCR Master Mix 4305719

2✕ Universal PCR Master Mix, No AmpErase® UNG, 200

2✕ Universal PCR Master Mix, No AmpErase® UNG, 2000

2✕ Universal PCR Master Mix, No AmpErase®

PCR Core Reagents Kit N808-0228

4369542

4324018

4326614

4324020

For More

Information

Note: If you purchase the TaqMan

with TaqMan

2✕ Universal PCR Master Mix kit (PN 4308320), you do not need to

Exogenous Internal Positive Control Reagents

purchase the master mix separately.

Note: Presence/absence experiments are not supported for Fast or SYBR

master mixes and protocols.

For information on using the TaqMan reagents, refer to the:

• TaqMan

Gene Expression Master Mix Protocol

Universal PCR Master Mix Protocol

Green

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Chapter 5 Presence/Absence Experiments

Design the Experiment

Use the StepOne

Software

For Applied Biosystems Inventoried/Made to Order assays, use the StepOne software to design your presence/absence experiments. The StepOne software automatically calculates volumes for the:

Note: To select the Inventoried/Made to Order assay type in the StepOne software,

go to the Reaction Setup screen in either the Design Wizard or Advanced Setup, then select Inventoried/Made to Order from the Assay Type dropdown menu.

For More

Information

For information on designing and performing presence/absence experiments on the StepOne and StepOnePlus systems, refer to the Applied Biosystems StepOne

StepOnePlus Experiments.

Custom Assays

Workflow

If you select the Custom assay type in the StepOne™ software for a presence/absence experiment (that is, you are designing your own primers and probes), Applied Biosystems recommends that you follow the workflow for the Applied Biosystems Assay Design Guidelines:

1. Design primers and probes using Primer Express

• Reaction mix components

• Controls and samples

• Sample dilutions

™

Real-Time PCR Systems Getting Started Guide for Presence/Absence

Software (page 3-21).

™

and

2. Select the appropriate reagents (page 3-24).

IMPORTANT! Applied Biosystems does not recommend the use of TAMRA

™

dye as a reporter or quencher with the StepOne system. TAMRA dye may be used as a reporter or quencher with the StepOnePlus system.

Presence/absence experiments are not supported for Fast or SYBR

Green

master mixes and protocols.

3. Use the recommended thermal cycling conditions (page 3-27).

4. Begin with default primer and probe concentrations. If needed, optimize the primer concentrations (page 3-30) and probe concentrations (page 3-33).

IMPORTANT! These steps provide a rapid and reliable system for assay design and

Note: To select the Custom assay type in the StepOne software, go to the Reaction

Setup screen in either the Design Wizard or Advanced Setup, then select Custom from the Assay Type dropdown menu.

5-16 Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems Reagent Guide