User guide

ManualsBrandsBio-Rad ManualsReceivers and AmplifiersSingleShot™ Cell Lysis RT-qPCR Kits

Introduction

The SingleShot Cell Lysis 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

Cell Lysis Kit, gene expression analysis can be completed in approximately 2 hours

from cell culture to quantiﬁcation cycle (Cq) using either a two-step or a one-step

RT-qPCR workﬂow.

Kit Contents

Cell Lysis Reagents 100 x 50 μl reactions 500 x 50 μl reactions

SingleShot Cell Lysis Buffer 5 ml (1 x 5 ml vial) 25 ml (5 x 5 ml vial)

Proteinase K Solution 100 µl (1 x 1 ml vial) 500 µl (1 x 1 ml vial)

DNase Solution 100 µl (1 x 1 ml vial) 500 µl (1 x 1 ml vial)

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

RT-qPCR Products Required but Not Provided

The SingleShot Cell Lysis Kit is validated for the products listed in Table 1.

Additional Reagents Required but Not Provided



Phosphate buffered saline (PBS) for washing the cells



TE buffer (nuclease-free) pH 7.5 for use with the PrimePCR Reverse Transcription

Control Assay

Table 1. Validated RT-qPCR products for use with SingleShot Cell Lysis Kit.

Catalog # Description

Two-Step RT-qPCR Products

172-5038 iScript

™

Advanced cDNA Synthesis Kit for RT-qPCR, 100 reactions

172-5271 SsoAdvanced

™

Universal SYBR

Green Supermix, 500 reactions

172-5281 SsoAdvanced Universal Probes Supermix, 500 reactions

One-Step RT-qPCR Products

172-5151 iTaq

™

Universal SYBR

Green One-Step Kit, 100 reactions

172-5141 iTaq

™

Universal Probes One-Step Kit, 100 reactions

Control (optional)

Online order only PrimePCR

™

Reverse Transcription Control Assay (bio-rad.com/primepcr)

For research purposes only.

Catalog # Description

172-5080 SingleShot Cell Lysis Kit, 100 x 50 µl reactions

172-5081 SingleShot Cell Lysis Kit, 500 x 50 µl reactions

SingleShot

™

Cell Lysis Kit

Summary of content (10 pages)

PAGE 1
SingleShot™ Cell Lysis Kit Catalog # Description 172-5080 172-5081 SingleShot Cell Lysis Kit, 100 x 50 µl reactions SingleShot Cell Lysis Kit, 500 x 50 µl reactions For research purposes only. Introduction The SingleShot Cell Lysis 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.
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™ SingleShot Cell Lysis 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.
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™ SingleShot Cell Lysis 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 3). Mix thoroughly and centrifuge. Use within 2 hr. Table 3. Preparation of SingleShot cell lysis master mix for nonadherent cells. Volume per Well, µl Volume for 96-Well Plate, µl SingleShot Cell Lysis Buffer 48 4,608 Proteinase K Solution 1 96 DNase Solution 1 96 Component 2. Count the cells.
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™ SingleShot Cell Lysis Kit Preparation of RT-qPCR Reactions For one-step and two-step RT-qPCR reactions using cell lysate prepared with the SingleShot Cell Lysis Kit, follow the instructions in Table 4 with respect to input lysate volume. Follow the respective instructions for thermal cycling and reaction volumes in the product inserts for the reagents listed in Table 4. Analyze the RT-qPCR data using standard methodologies. Table 4. Input volume of SingleShot cell lysate for RT-qPCR reactions.
PAGE 5
™ SingleShot Cell Lysis Kit Table 5. Preparation of SingleShot cell lysis master mix containing the RNA control. Reagent SingleShot Cell Lysis Buffer Reagent per 4-log Tenfold Dilution Series in a 96-Well Plate, µl 235 Proteinase K Solution 5 DNase Solution 5 RNA Control Template Total volume 5 250 4. Select input lysate volumes for the RT-qPCR reactions following the recommendations in Table 4. Maintain the same lysate and cDNA volumes for all reactions in this experiment.
PAGE 6
™ SingleShot Cell Lysis Kit 40 Target 1 Target 2 35 Control RNA Cq 30 25 20 15 0 1 2 3 Cell number, log 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. Using the PrimePCR Reverse Transcription 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.
PAGE 7
™ SingleShot Cell Lysis Kit 6. Perform data analysis according to the following guidelines: ■■ ■■ NA control: Plot the Cq values for the RNA control against the percentage input R lysate (Figures 2A and 2B). A constant Cq value across the input lysate range indicates no RT-qPCR inhibition. A deviation of >1 Cq value indicates RT-qPCR inhibition.
PAGE 8
™ SingleShot Cell Lysis Kit Appendix A Table 1. Setting up SingleShot cell lysis reactions. Step Preparing the cell culture Washing the cells with PBS Preparing the SingleShot cell lysis master mix Guidelines Number of wells 384 96 48 24 12 6 Cell numbers per well at harvest 10– 5 x 104 10– 1 x 105 10– 2 x 105 10– 4 x 105 10– 8 x 105 10– 1 x 10 6 Volume of PBS per well, µl 30 125 250 500 1,000 2,000 SingleShot Cell Lysis Buffer, µl 12 48 96 192 384 768 Proteinase K, µl 0.
PAGE 9
™ SingleShot Cell Lysis Kit Troubleshooting Guide Problem No amplification in the RT-qPCR reaction Delayed Cq values seen in RNA detection Potential Cause ■ ■ ■ ■ Cell lines may contain high levels of PCR inhibitors Solution ■ 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 ■ ■ ■ ■ ■ Genomic DNA is amplified as seen in the no-RT control Signal in no template control (NTC) reaction ■ ■ ■ ■
PAGE 10
™ SingleShot Cell Lysis Kit Visit bio-rad.com/web/SSCellLysis for more information. SYBR is a trademark 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. Purchase of iTaq and SsoAdvanced products 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.