GeneCopoeia AORT-0020, AORT-0050 User Guide

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All‐in‐One

Forreliablefirst‐strandcDNAsynthesisfromallRNAsources

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Cat.No.AORT‐0020(20synthesisreactions)
Cat.No.AORT‐0050(50synthesisreactions)

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UserManualI

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™

First‐StrandcDNASynthesisKit

GeneCopoeia,Inc.
9620MedicalCenterDrive,#101
Rockville,MD20850
USA

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301‐762‐0888
866‐360‐9531


[e-mail]

www.genecopoeia.com

© 2009 GeneCopoeia, Inc.

All-in-One™ First-Strand cDNA Synthesis Kit

USER MANUAL I

™

All-in-One

I. Description
II. Related Products
III. Contents and Storage
IV. Preparation
V. Procedure
VI. Example
VII. Trouble Shooting Guide
VIII. Limited Use License and Warranty

I. Description

The All-in-One™ First-Strand cDNA Synthesis Kit includes a reverse transcriptase and a specialized set of
reagents designed to yield first-strand cDNA that is optimal for gene cloning, cDNA library creation and
quantitative PCR amplification. A robust experimental design delivers a universal kit that is suitable for first­strand cDNA synthesis from almost any source of RNA.

The kit uses Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus (M-MLV RTase (H–))
which is an RNA-dependent DNA polymerase used in cDNA synthesis with long RNA templates (>13kb).
The lack of RNase H activity is important in this application in that RNase H activity will start to degrade
template during long incubation times required for producing long cDNAs. RNase H minus RT enables
preparation of long cDNAs and libraries containing a high percentage of full-length cDNA.

II. Related Products

First-Strand cDNA Synthesis Kit

GeneCopoeia Description

All-in-One™ qPCR Mix

All-in-One™ qPCR Primers

All-in-One™ qPCR Primer Array

All-in-One™ miRNA qRT-PCR
Detection Kits

All-in-One™ miRNA qPCR Primers

OmicsLink™ Expression-Ready
ORF cDNA Clones

Endofectin™ Transfection Reagents

SYBR Green-based real-time quantitative
PCR Mix

Human, mouse and rat
primers

User specified, ready-to­use primer arrays in 96­well-plate format

®

Green-based Accurately quantify miRNA expression

SYBR

Human, mouse and rat
primers

20,000 human
15,000 mouse

Optimized for specific cell
types

Validated, gene-specific primers ensure
specificity and sensitivity

Reliable tools ideal for analyzing the
expression of a focused panel of genes
such as pathways, diseases or customized
gene panels

Validated for robust, reproducible and
reliable quantitation of miRNA activity

Perform a variety of applications with
expression-ready clones

Transfect efficiently and with low toxicity

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All-in-One™ First-Strand cDNA Synthesis Kit

III. Contents and Storage

Contents and storage recommendations for the All-in-One First-Strand cDNA Synthesis Kit

Cat. Nos. AORT-0020 and AORT-0050

Contents Quantity Storage temperature/ conditions

200 U/µl M-MLV Reverse
Transcriptase (RNase H-)

5X RT Reaction Buffer

25 U/µl RNase Inhibitor

are provided in the following table.

–20°C (Stable for at least 12 months)

1 x 20 µl

1 x 50 µl

1 x 100 µl

1 x 250 µl

1 x 20 µl

1 x 50 µl

Alternatively, the solution can also be

stored at -80°C in aliquots. Avoid

repeated freezing/ thawing.

–20°C (Stable for at least 12 months)

Alternatively, the solution can also be

stored at -80°C in aliquots. Avoid

repeated freezing/ thawing.

–20°C (Stable for at least 12 months)

Alternatively, the solution can also be

stored at -80°C in aliquots. Avoid

repeated freezing/ thawing.

25 mM dNTP

60 µM Oligo (dT)18

250 µM Random Primer

dd H2O (RNase and DNase free)

1 x 20 µl

1 x 50 µl

1 x 20 µl

1 x 50 µl

1 x 20 µl

1 x 50 µl

1 x 1 ml

1 x 1 ml

–20°C (Stable for at least 12 months)

Alternatively, the solution can also be

stored at -80°C in aliquots. Avoid

repeated freezing/ thawing.

–20°C (Stable for at least 12 months)

Alternatively, the solution can also be

stored at -80°C in aliquots. Avoid

repeated freezing/ thawing.

–20°C (Stable for at least 12 months)

Alternatively, the solution can also be

stored at -80°C in aliquots. Avoid

repeated freezing/ thawing.

–20°C (Stable for at least 12 months)

Alternatively, the solution can also be

stored at -80°C in aliquots. Avoid

repeated freezing/ thawing.

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All-in-One™ First-Strand cDNA Synthesis Kit

IV. Preparation

Wearing a lab coat, disposable gloves and protective goggles are recommended when handling
chemicals.

RNA Sample Preparation

When working with RNA it is important to avoid RNases in your solutions, consumables and labware. When
preparing your RNA samples, always wear a mask and disposable gloves in all procedures. Follow the
described procedures you are using for RNA extraction carefully. Ready-to-use solutions that are RNase­free can be purchased. Alternatively treat solutions with diethyl pyrocarbonate (DEPC) and then autoclave.
RNases on labware can also be inactivated by DEPC treatment or by baking at 250°C for 3 hours. Use
DEPC to treat all microcentrifuge tubes, pipettes and pipette tips (if not RNase free) and then autoclave to
deactivate RNases. RNase-free consumables are available for purchase from many commercial sources.

IMPORTANT NOTES:

1. Store kit at –20°C. Avoid storage or leaving reagents at 4°C or room temperature.

2. Mix reagents thoroughly by gently inverting tubes several times avoiding bubbles and then briefly
centrifuge before use.

3. Set up all reactions on ice to reduce risk of RNA degradation.

4. Read all procedures before setting up RT reaction

V. Procedure

1. Thaw all the reagents needed for RNA reverse transcription from the First-Stand cDNA Synthesis
Kit. Mix reagents well by gently inverting the tubes. Spin down briefly and keep on ice.

2. Prepare the RNA-Primer Mix: Add the following reagents into an RNase-free reaction tube which
has been pre-cooled on ice. The final volume should be 13μl.

Reagents Volume Final concentration

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Total RNA

or polyA RNA

250 μM Random Primer

or 60 µM Oligo (dT)

or 10 µM sequence-specific primer

ddH2O (RNase/DNase free) to 13 μl

†

The amount of RNA in the table is the recommended amount. The total RNA may be adjusted to between 10 ng ~ 5 µg

and the purified poly A RNA between 1 ng ~ 100 ng.

††

Please choose one of the RT Primers based on the experimental design. The reverse transcription will begin at the

polyA tail if using the Oligo (dT)18. It will begin at many different RT sites throughout the RNA if using the Random
Primer.

18

1 μl

1 μg

or 10 ng

10 μM

or 2.4μM

or 0.4 μM ††

†

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All-in-One™ First-Strand cDNA Synthesis Kit

3. Denature RNA: Mix the reaction solution well. Spin down briefly. Heat the RNA-Primer mix at 65°C
for 10 minutes, then cool it down immediately on ice.

4. Prepare RNA reverse transcription reaction: Add the following reagents into the RNA-Primer mix
reaction tube which has been cooled on ice. The final volume should be 25 μl.

Reagents Volume Final concentration

RNA-Primer Mix 13 μl

5X RT Reaction Buffer 5 μl 1X

25 mM dNTP

25 U/μl RNase Inhibitor

200 U/μl M-MLV RTase

ddH2O (RNase/DNase-free) to 25 μl

5. Reverse Transcription Reaction: Mix reaction solution well. Spin down briefly. Incubate the reaction
solution at 37°C for 60 minutes if using the Random Primer, or incubate at 42°C for 60 minutes if
using the oligo (dT)

6. Terminate the reaction by heating at 85°C for 5 minutes and then store at –20°C.

7. The cDNA reaction product can be used directly in the next step without being purified. A volume of

0.5

μl ~ 2 μl of undiluted cDNA is recommended for standard 25 μl PCR reactions. If performing

quantitative PCR, it is recommended to do a 1:5 ~ 1:20 dilution of the cDNA and add a volume of 2

or sequence-specific primer.

18

1 μl 1 mM

1 μl 1 U

1 μl 8 U

μl for each 20-μl qPCR reaction.

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All-in-One™ First-Strand cDNA Synthesis Kit

VI. Example

Objective: The reverse transcription efficiency of the All-in-One First Strand Synthesis Kit is assessed by

examining the amplification results of different genes or gene regions using the oligo(dT) synthesized cDNA
prepared from the All-in-One First-Strand cDNA Kit.

Figure 1. Efficient cDNA synthesis by All-in-One™ First-Strand cDNA Synthesis Kit. Total RNA

isolated from human placenta was used as template RNA in reverse transcription reactions using

the All-in-One First-Strand cDNA Synthesis Kit together with the oligo(dT) primer. The synthesized

cDNA was then used to amplify different gene regions by quantitative PCR using the All-in-One

qPCR Mix (GeneCopoeia Catalog No. AOPR-0200). The positive amplification results of MACF1

indicate that up to a 13 kb RNA sequence was reversed transcribed.

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All-in-One™ First-Strand cDNA Synthesis Kit

VII. Trouble Shooting Guide

RNA template degradation

Little or no RT-PCR
product

PCR product is longer
than expected

• The quality of the RNA is the key factor for cDNA synthesis. Follow

the RNA isolation kit procedure carefully, always wearing a lab coat,

gloves and mask when working with RNA and use RNA-Grade

reagents and materials. Check the RNA quality by RNA

electrophoresis in a denaturing gel.

An inhibitor was present in the RNA template

• Trace amounts of inhibitor such as guanidine salts in the RNA

template can inhibit the cDNA synthesis. Re-precipitate the RNA with

ethanol and wash the pellet with 75% ethanol.

A G-C rich template or secondary structure of the amplification
product is obstructing the reaction

• Prepare the RNA-Primer Mix before the RT step. Then add a PCR

enhancing reagent such as DMSO, betaine, etc. in the PCR reaction.

• Genomic DNA was present. Perform a DNase I digest before the RT

step or design intron-spanning or flanking primers to avoid co-

amplification of genomic DNA.

• The wrong product was amplified. Optimize the PCR reaction

conditions.

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All-in-One™ First-Strand cDNA Synthesis Kit

VIII. Limited Use License and Warranty

Limited Use License

Following terms and conditions apply to use of all OmicsLink™ ORF Expression Clones in all lentiviral vectors and Packaging Kit (the
Product). If the terms and conditions are not acceptable, the Product in its entirety must be returned to GeneCopoeia within 5 calendar days.
A limited End-User license is granted to the purchaser of the Product. The Product shall be used by the purchaser for internal research
purposes only. The Product is expressly not designed, intended, or warranted for use in humans or for therapeutic or diagnostic use. The
Product must not be resold, repackaged or modified for resale, or used to manufacture commercial products without prior written consent
from GeneCopoeia. This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic
research. Use of any part of the Product constitutes acceptance of the above terms.

Limited Warranty

GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet. If it is proven to the
satisfaction of GeneCopoeia that the Product fails to meet these specifications, GeneCopoeia will replace the Product. In the event a
replacement cannot be provided, GeneCopoeia will provide the purchaser with a refund. This limited warranty shall not extend to anyone
other than the original purchaser of the Product. Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt
of the Product. GeneCopoeia’s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price.
GeneCopoeia’s liability does not extend to any damages arising from use or improper use of the Product, or losses associated with the use
of additional materials or reagents. This limited warranty is the sole and exclusive warranty. GeneCopoeia does not provide any other
warranties of any kind, expressed or implied, including the merchantability or fitness of the Product for a particular purpose.

GeneCopoeia is committed to providing our customers with high-quality products. If you should have any questions or
concerns about any GeneCopoeia products, please contact us at 301-762-0888.

© 2009, GeneCopoeia, Inc.

GeneCopoeia, Inc.
9620 Medical Center Drive
Rockville, MD 20850
Tel: [phone] Fax: [phone]
Email:
Web:

[e-mail]

HUwww.genecopoeia.comU

GeneCopoeia Products are for Research Use Only Copyright © 2010 GeneCopoeia, Inc.
Trademarks: GeneCopoeia™, OmicsLink™, All-in-One™, EndoFectin™ (GeneCopoeia Inc.);

®

SYBR

(Molecular Probes); iQ™5 (Bio-Rad); ROX® (Invitrogen). AOFS1-0210

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