Bio-Rad SingleShot Cell Lysis RT-qPCR Kits User Manual

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SingleShot™ SYBR® Green Kit

Catalog # Description

172-5 0 8 5 SingleShot

™

SYBR® Green Kit, 100 x 50 µl reactions

For research purposes only.

Introduction

The SingleShot

™

SYBR® Green Kit prepares genomic DNA (gDNA)–free RNA directly from cell culture in approximately 20 min for use in reverse transcription quantitative PCR (RT-qPCR) applications. This kit is compatible with an input of 100,000–10 cells from suspension, adherent cells, or primary cells from cell cultures. With the SingleShot

™

SYBR® Green Kit, gene expression analysis can be completed in approximately 2 hours from cell culture to quantiﬁcation cycle (Cq). This kit includes reagents for the RT-qPCR reactions and a SYBR

Green–based qPCR control assay

to optimize input cell number and input lysate amount.

Kit Contents (100 x 50 µl reactions)

Cell Lysis Reagents Description

SingleShot Cell Lysis Buf fer 5 ml (1 x 5 ml vial) Proteinase K Solution 100 µl (1 x 1 ml vial) DNase Solution 100 µl (1 x 1 ml vial)

Store all compone nts at –20°C for up to 1 year.

Controls

SingleShot RNA Control 200 reactions SingleShot RNA Control Template Lyophilized SingleShot

Store the control template, once resuspended, at – 80°C. Store the qPCR assay at –20°C for up to 1 year.

RT-qPCR Reagents

iScript 5x iScript Advanced Reaction Mix 1 vial iScript Advanced Reverse Transcriptase 1 vial Nuclease-Free Water 1.5 ml SsoAdvanced

Store the RT-qPCR products at –20°C for up to 1 year. The SsoAdvanced Supermix can be stored at 4°C for up to 3 months.

™

SYBR® Green qPCR Control Assay 200 µl

™

Advanced cDNA Synthesis Kit for RT-qPCR 100 reactions

™

Universal SYBR® Green Supermix 5 x 1 ml vial (500 reactions)

Reagents Required but Not Provided



Phosphate buffered saline (PBS) for washing the cells



TE buffer (nuclease-free) pH 7.5 for resuspending the SingleShot RNA control template

SingleShot™ SYBR® Green Kit

Processing of Adherent Cells in a 96-well Culture Plate

For processing adherent cells in non–96-well cell culture plates, refer to Table 1

in Appendix A

For processing trypsinized adherent cells, neutralize the trypsin with culture

medium. Follow instructions in Processing Nonadherent Cells in a 96-Well PCR Plate section

1. Seed the cell culture in advance in a 96-well culture plate so that the cell numbers

at harvest are in the range of 100,000–10 cells/well.

For adherent cells, it is important to use cells that are fully adhered to the plate

to avoid cell loss during washing

Using too many cells may result in incomplete cell lysis and can inhibit RT-qPCR.

For optimal results, we recommend using the SingleShot RNA control included in this kit to determine the appropriate input cell number

2. Prepare fresh on ice the appropriate volume of SingleShot cell lysis master mix

(see Table 1). Mix thoroughly and centrifuge. Use within 2 hr.

Table 1. Preparation of Sing leShot cell lysis master mix for adherent cells.

Component

SingleShot Cell Lysis Buf fer

Proteinase K Solution 1 96

DNase Solution 1 96

Volume per

Well, µ l

48 4,608

Volume for 96-Well

Plate, µl

3. Remove cell culture medium completely by aspiration.

4. Wash cells with 125 μl of room temperature PBS. Aspirate to remove

PBS completely.

5. Add 50 μl of SingleShot cell lysis master mix to each well.

6. Incubate without agitation for 10 min at room temperature.

Do not mix the cells with the solution by pipetting. For step 6, do not exceed

20 min at room temperature

7. Transfer the cell lysate to a PCR plate or centrifuge tube. Incubate at 37°C for

5 min, followed by 5 min at 75°C.

Use a thermal cycler for best thermal uniformity

8. The cell lysate can be stored for up to 4 hr on ice, for up to 2 months at –20°C,

or for up to 12 months at –80°C.

9. Go to the Preparation of Reverse Transcription Reactions section.

SingleShot™ SYBR® Green Kit

Processing of Nonadherent Cells in a 96-Well PCR Plate

1. Prepare fresh on ice the appropriate volume of SingleShot cell lysis master mix

(Table 2). Mix thoroughly and centrifuge. Use within 2 hr.

Table 2. Preparation of SingleShot cell l ysis m aste r mix for nonadherent cells.

Component

SingleShot Cell Lysis Buf fer

Proteinase K Solution 1 96

DNase Solution 1 96

Volume per

Well, µ l

48 4,608

Volume for 96-Well

Plate, µl

2. Count the cells. Transfer appropriate number of cells (10–10

cells per well) to a

96-well PCR plate or tube.

3. Centrifuge at 500–1,000 x g for 5 min. Remove as much of the medium as possible

without disturbing the cell pellet.

4. Wash cells with 125 μl room temperature PBS. Centrifuge at 500–1,000 x g

for 5 min. Carefully remove 120 μl of the supernatant using a pipet, leaving approximately 5 μl PBS in each well.

5. Add 50 μl of SingleShot cell lysis master mix to each well. Pipet up and down

5 times to ensure complete resuspension of the cell pellet.

6. Incubate for 10 min at room temperature, followed by 5 min at 37°C, and 5 min

at 75°C.

7. The cell lysate can be stored on ice for up to 4 hr, at –20°C for up to 2 months,

or at –80°C for up to 12 months.

8. Go to the Preparation of Reverse Transcription Reactions section.

SingleShot™ SYBR® Green Kit

Preparation of Reverse Transcription Reactions

1. For optimal results, reactions should be assembled on ice. Prepare the reverse

transcription reaction according to the directions in Table 3. Mix thoroughly by pipetting up and down several times.

Table 3. Preparation of reverse transcription reaction.

Component Volume per 20 μl Reaction, µl

5x iScript Advanced Reaction Mix 4

iScript Advanced Reverse Transcriptase 1

Cell Lysate 4–9

Nuclease-Free Water Variable

Total reaction mix volume 20

2. Incubate the complete reaction mix in a thermal cycler using the following protocol:

reverse transcription for 30 min at 42°C followed by RT inactivation for 5 min at 85°C.

3. Proceed to the Preparation of qPCR Reactions section, or store the cDNA

at –20°C.

Recommendations for the use of no-RT control



Interference of gene expression analysis by gDNA carryover in cell lysate samples can be tested by setting up a no-RT control reaction



The reverse transcriptase volume in a no-RT control reaction should be replaced with water



The same amount of cell lysate used in the RT reaction should be used in the no-RT reaction to ensure similar carryover of cDNA synthesis components in a qPCR reaction

SingleShot™ SYBR® Green Kit

Preparation of qPCR Reactions

Instrument Compatibility

SsoAdvanced™ Universal SYBR® Green Supermix is compatible with all Bio-Rad and other commercially available real-time PCR systems.

Reaction Mix Preparation and Thermal Cycling Protocol

1. Thaw SsoAdvanced™ Universal SYBR® Green Supermix and other frozen reaction

components to room temperature. Mix thoroughly, centrifuge brieﬂy to collect solutions at the bottom of tubes, then store on ice protected from light.

2. Prepare (on ice or at room temperature) enough qPCR reaction mix for all qPCR

reactions by adding all required components, except the template, according to the recommendations in Table 4.

Table 4. qPCR reaction setup.*

Component

SsoAdvanced™ Universal SYBR® Green Supermix (2x)

Forward and Reverse Primers Variable Variable 250 –500 nM each primer

cDNA (add at step 4)

Nuclease-Free Water Variable Variable —

Total reaction mix volume 10 20 —

* Scale all components proportionally according to sample number and reaction volumes.

Volume per 10 µl

Reaction, µl

5 10 1x

1–2 2–4 —

Volume per 20 µl

Reactions, µl

Final

Concentration

3. Mix the qPCR reaction mix thoroughly to ensure homogeneity and dispense equal

aliquots into each PCR tube or into the wells of a PCR plate. Use good pipetting technique to ensure assay precision and accuracy.

4. Add cDNA (prepared in the Preparation of Reverse Transcription Reactions section)

to the PCR tubes or wells containing qPCR reaction mix (prepared according to Table 4), seal tubes or wells with ﬂat caps or optically transparent ﬁlm, and vortex 30 sec or more to ensure thorough mixing of the reaction components. Spin the tubes or plate to remove any air bubbles and to collect the reaction mixture in the vessel bottom.

SingleShot™ SYBR® Green Kit

Table 5. Thermal cycling protocol.

Real-Time PCR System

Bio-Rad® CFX96™,

™

, CFX96 Touch™,

CFX384 CFX96 Touch Deep Well, CFX384 Touch CFX Connect

Bio-Rad® iQ™5, MiniOpticon MyiQ

Applied Biosystems 7500 and 7900 HT, QuantStudio

Roche LightCycler 96 or 480

QIAGEN Rotor-Gene and Stratagene Mx series

* Shorter annealing/extension times (1–10 sec) can be used for amplicons <100 bp. Longer annealing/

extension times (30– 60 sec or more) can be used for amplicons >250 bp, GC- or AT- rich targets, low-expressing targets, crude samples, or higher input amounts (for example, 4 µl of cDNA).

™

, Chromo4™,

Setting/ Scan

Mode

only

SYBR

Standard 15–3 0

Standard

Fast

Standard 60

Fast

Polymerase Activation and

DNA Denaturation

30 sec at

98°C for cDNA

5. Program the thermal cycling protocol on a real-time PCR instrument according

to Table 5.

6. Load the PCR tubes or plate into the real-time PCR instrument and start the

PCR run.

7. Perform data analysis according to the instrument-speciﬁc instructions.

Recommendations for assay design and optimization



For best qPCR efﬁciency, design assays targeting an amplicon size of 70–150 bp



The SsoAdvanced™ Universal SYBR® Green Supermix and the recommended qPCR cycling protocols have been optimized for assays with a primer melting temperature (T

) of 60°C designed using the open source Primer3, Primer3Plus,

or Primer-BLAST programs at their default settings. If primers are designed using other programs, adjust the annealing temperature accordingly

Ampliﬁcation

SingleShot™ SYBR® Green Kit

Denaturation

at 95°C, sec

5–15

Annealing/Extension and Plate Read

at 60°C, sec* Cycles Melt Curve Analysis

15–3 0

10–3 0

15–3 0

35–40

65–98°C

0.5°C increments at 2–5 sec/step

(or use instrument

default setting)

SingleShot™ SYBR® Green Kit

Optimizing Input Cell Number and Input Lysate Amount

For best results, the SingleShot RNA control can be used to determine optimal input cell number and optimal input lysate volume.

The SingleShot RNA control includes a synthetic RNA template that has no homology to any known sequence and a qPCR assay speciﬁc for this RNA template.

The template RNA is shipped lyophilized. Upon resuspension, store at –80°C.

Using the SingleShot RNA Control Assay to Determine Optimal Input Cell Number

To determine optimal input cell number, adherent cells must be trypsinized for

accurate cell counting

1. Resuspend the RNA control template in 200 µl of nuclease-free TE buffer pH 7.5.

2. Prepare a tenfold serial dilution of 100,000–10 cells in PBS.

3. Prepare the SingleShot cell lysis master mix according to the directions in Table 6.

Table 6. Preparation of SingleShot cell lysis master mix for optimizing input cell number.

Reagent

SingleShot Cell Lysis Buf fer 235

Proteinase K Solution 5

DNase Solution 5

SingleShot RNA Control Template 5

Total volume 250

Reagent per 4-log Tenfold D ilution Series

in a 96-Well Plate, µl

4. Perform the reverse transcription reactions following the previously mentioned protocol.

5. Prepare the qPCR reaction following recommendations in Table 7. Do not add template until step 8. Maintain the same input lysate, cDNA, and qPCR volumes for all reactions in this experiment.

If desired, gene expression targets of interest can be ampliﬁed in parallel with

the RNA control assay

Table 7. Preparation of qPCR reaction mix fo r RNA control assay.*

Component

SsoAdvanced Green Supermix (2x)

SingleShot qPCR Control Assay

cDNA (add at step 8)

Nuclease-Free Water Variable Variable —

Total reaction mix volume 10 20 —

* Scale all components proportionally according to sample number and reaction volumes.

™

Universal SYBR®

™

SYBR® Green

Volume per 10 µl

Reaction, µl

5 10 1x

0.5 1 1x

1–2 2–4 —

Volume per 20 µl

Reactions, µl

Final

Concentration

SingleShot™ SYBR® Green Kit

6. Mix the qPCR reaction mix thoroughly to ensure homogeneity and dispense equal aliquots into each PCR tube or into the wells of a PCR plate. Use good pipetting technique to ensure assay precision and accuracy.

7. Add cDNA to the PCR tubes or wells containing qPCR reaction mix (prepared using Table 7), seal tubes or wells with ﬂat caps or optically transparent ﬁlm, and gently vortex to ensure thorough mixing of the reaction components. Spin the tubes or plate to remove any air bubbles and to collect the reaction mixture in the vessel bottom.

8. Program the thermal cycling protocol on a real-time PCR instrument according to Table 5.

9. Perform data analysis according to the following guidelines:



RNA control: Plot the Cq values for the RNA control against the log of the number of cells used to generate the lysate (Figure 1). A constant Cq value across the input cell range indicates complete lysis and no RT-qPCR inhibition. A deviation of >1 Cq value indicates incomplete lysis and/or RT-qPCR inhibition. Input cell numbers that show such a Cq deviation should be avoided. In the example shown in Figure 1, optimal performance can be achieved with 100,000–10 input cells



Target gene: Plot the Cq values for the target gene against the log of the number of cells used to generate the lysate (Figure 1). A decrease in Cq value is expected as cell number increases. The decrease in Cq values should be linear for cell numbers that don’t exhibit inhibitory effects. Deviation from linearity results from incomplete lysis and/or RT-qPCR inhibition

0 1 2 3 4 5

Fig. 1. Determining optimal cell input number. In this example, 105 input cells is the maximum input. No inhibition was noted across the input series. Target genes demonstrate linearity across all four logs.

Cell nu mber, log

Target 1

Tar get 2

Control RNA

SingleShot™ SYBR® Green Kit

Using the SingleShot RNA Control Assay to Determine Optimal Input Lysate Volume

1. Resuspend the RNA control template in 200 µl of nuclease-free TE buffer pH 7.5.

2. Prepare cell lysate from either adherent (see Processing of Adherent Cells in a 96-Well Culture Plate section) or suspension cells (see Processing of Nonadherent Cells in a 96-Well PCR Plate section) with an optimal number of input cells.

3. Vary input cell lysate in the reverse transcription reactions as shown in Table 8.

Table 8. Setup of reverse transcription reactions to optimize input lysate amount.

Input Lysate, %

10 2 1 10 7

20 4 1 10 5

30 6 1 10 3

40 8 1 10 1

45 9 1 10 0

* Includes 5x iScript advanced reaction mix.

Lysa te

Volume, µl

RNA Control Template, μl

2x RT Master

Mix,* µl

Nuclease-Free

H2O, µl

4. Incubate the complete reaction mix in a thermal cycler using the following protocol: reverse transcription for 30 min at 42°C followed by RT inactivation for 5 min at 85°C.

5. Set up qPCR reactions following instructions in Table 9. Do not add cDNA until step 7.

Table 9. Preparation of qPCR reaction mix to determine optimal input lysate amount.*

Component Volume per 20 µl Reactions, µl Final Concentration

SsoAdvanced Green Supermix (2x)

SingleShot Control Assay

cDNA (add at step 7)

Nuclease-Free Water Variable —

Total reaction mix volume 20 —

* Scale all components proportionally according to sample number and reaction volumes.

™

Universal SYBR®

™

SYBR® Green qPCR

10 1x

1 1x

7 —

7. Add cDNA to the PCR tubes or wells containing qPCR reaction mix (prepared using Table 9), seal tubes or wells with ﬂat caps or optically transparent ﬁlm, and gently vortex to ensure thorough mixing of the reaction components. Spin the tubes or plate to remove any air bubbles and to collect the reaction mixture in the vessel bottom.

SingleShot™ SYBR® Green Kit

8. Program the thermal cycling protocol on a real-time PCR instrument according to Table 5.

9. Perform data analysis according to the following guidelines:



RNA control: Plot the Cq values for the RNA control against the percentage input lysate (Figure 2). A constant Cq value across the input lysate range indicates no RT-qPCR inhibition. A deviation of >1 Cq value indicates RT-qPCR inhibition. Input lysate amounts that show such a Cq deviation should be avoided. In the example shown in Figure 2, optimal performance can be achieved with input lysate amounts up to 45%.



Target gene: If a target gene is ampliﬁed in parallel with the RNA control, plot the Cq values for the target gene against the percentage input lysate (Figure 2). A decrease in Cq value is expected as input lysate increases. Deviation in the linear response of the RNA control results from incomplete lysis and/or RT-qPCR inhibition.

10 20 30 40 45

Fig. 2. Determining optimal input cell lysate amounts. The maximum allowable cell lysate input is 45%.

Input lysate, %

Tar get

Control RNA

SingleShot™ SYBR® Green Kit

Appendix A

Table 1. Set ting up Sin gleShot cell lysis reactions.

Step Guidelines

Preparing the cell culture

Washing the cells with PBS

Preparing the SingleShot cell lysis master mix

Number of wells 384 96 48 24 12 6

Cell numbers per well at harvest

Volume of PBS per well, µl

SingleShot Cell Lysis Buffer, µl

10–

5 x 10

30 125 250 500 1,000 2,000

12 48 96 192 384 76 8

10–

1 x 10

2 x 10

Proteinase K, µl 0.25 1 2 4 8 16

DNase, µl 0.25 1 2 4 8 16

Total volume per well, µl

12. 5 50 100 200 400 800

10–

4 x 10

10–

8 x 10

10–

1 x 10

Troubleshooting Guide

Problem Potential Cause Solution

No ampliﬁcation in the RT-qPCR reaction

Delayed Cq values seen in RNA detection

Genomic DNA is ampliﬁed as seen in the no-RT control

Signal in no template control (NTC) reac tion



Cell lines may contain high

levels of PCR inhibitors



E xcess number of cells

used in the lysis reaction



E xcess cell culture

medium carryover



E xcess lysate used in

the RT-qPCR reaction



Incomplete gDNA

digestion



DNase and Proteinase K

were not added to the lysis reaction



DNA contamination

(NTC melt peak T identical to the target gene melt peak T



 Primer dimers (NTC melt

)

peak is broad with a T ~65 –75°C)



Depending on the cell species or culture conditions,

the number of cells or percentage lysate may require optimization (see Optimizing Input Cell Number and Input Lysate Amount section)



Generally ≤105 cells can be used successfully in

the SingleShot procedure, but if RT or qPCR fails, try using 5- to 10-fold fewer cells



Wash cells with PBS to remove contaminants in the

culture medium



Remove as much of the culture medium and PBS

as possible



Use a freshly prepared SingleShot cell lysis master

mix; keep on ice and use within 2 hr



Make sure DNase and proteinase K are added in

the SingleShot cell lysis master mix before treating the cells



Repeat the lysis step. Ensure DNase is added,

the thermal cycling conditions are correct, and the thermal cycler is working properly



E xamine the work ﬂow to identify potential

contamination sources by replacing reagents one by one; use new aliquots of assays



Evaluate the assay design for pr imer dimer formation;

perform gradient PCR to optimize the a nnealing temperature; use a primer matrix to determine the

optimal primer concentration

SingleShot™ SYBR® Green Kit

Visit bio-rad.com/web/SSSYBRG for more information.

LightC ycler is a trademark of Roche Diagnostics G mbH. Mx is a trademark of Stratagene Corporation. Rotor-Gene is a trademark of QIAGEN GmbH. StepOne and StepOnePlus are trademarks of Applied Biosystems. QuantStudio, SYBR, and ViiA are trademarks of Life Technologies Corporation. Bio-Rad Laboratories, Inc. is licensed by Life Technologies Corporation to sell reagents containing SYBR Green I for use in real-time PCR, for research purposes only.

Bio-Rad’s real-time thermal cyclers are covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367.

Purchase of SsoAdvanced Universal SYBR Green Supermix includes an immunity from suit under patents speciﬁed in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

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Bio-Rad SingleShot Cell Lysis RT-qPCR Kits User Manual

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