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

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.

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SYBR® Green qPCR Control Assay 200 µl

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Advanced cDNA Synthesis Kit for RT-qPCR 100 reactions

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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

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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.

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