SingleShot™ Cell Lysis RT-qPCR Kits

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Single Shot Cell Lysis RT-qPCR Kits

Overview

Gene Expression Data from Cell Lysates and Purified RNA

SingleShot™ Probes One-Step Kit lysates and purified RNA yield comparable gene expression results

SingleShot™ Probes One-Step Kit demonstrated superior correlation with an R2 of 0.9954 compared to the same samples, but purified using a column-based method. FastLane Cell Probe Kit from Qiagen demonstrated a less than optimal correlation with an R2 of 0.9307 compared to the same samples, but purified using a column-based method.
SingleShot Probes One-Step Kit (Bio-Rad) Cq values show high correlation (R2 = 0.9954) with Cq values generated from purified RNA. FastLane Cell Probe Kit (QIAGEN) lysates Cq values demonstrate less than optimal correlation (R2 = 0.9307) with Cq values generated from purified RNA.

Gene expression studies conducted using different sample preparation methods should yield the same relative results. The expression of five genes was assessed in a tenfold dilution series spanning 10–105 input cells to compare the use of lysate vs. purified RNA as input for real-time PCR. Cq, quantification cycle.

The SingleShot™ Cell Lysis Kit Shows Superior RNase Inhibition

Fluorescence detection of RNases

Degraded RNA transcripts may lead to less than optimal qPCR data. This assay detected the presence of RNases via fluorescence detection after the lysis solution was added but prior to heat treatment.

Fluorescence detection of RNases

The lysis solution in Bio-Rad’s SingleShot Cell Lysis Kit contains a potent RNase inhibitor, which preserves RNA integrity. Life Technologies’ RNaseAlert QC System Assay showed the presence of high levels of RNases in the FastLane Cell cDNA kit, resulting in RNA degradation. A water control was added for baseline determination. RFU, relative fluorescence units.

The SingleShot™ SYBR® Green Kit Efficiently Removes Genomic DNA

104–105 input cells were evaluated for gDNA carryover after DNase treatment in three different cell lines (HeLa, NCI-H1975, and NIH 3T3).

The SingleShot Kit completely removed gDNA. No amplification (Cq >40) was detected from the DNA control assay regardless of the starting amount of input cells.

The competitor kit amplified gDNA at high levels across all three cell lines at all starting amounts of input cells, which can cause inaccurate gene expression data because both cDNA and gDNA would be amplified.

Accurate gene expression studies are based on relative expression levels of RNA transcripts

Cq, quantification cycle; NCI, National Cancer Institute; NIH, National Institutes of Health.

Reproducibility of Technical Lysate Replicates Ensures Optimal Data

The SingleShot™ SYBR® Green Kit demonstrates high reproducibility between lysate replicates, indicated by a Pearson correlation of 0.99.

Two technical lysate replicates were examined across 4 neuroblastoma cell lines (SH-EP, SK-N-AS, NGP, IMR-32) using 10 SYBR® Green qPCR assays to evaluate the reproducibility of the SingleShot SYBR® Green Kit

Two technical lysate replicates of four neuroblastoma cell lines (SH-EP, SK-N-AS, NGP, and IMR-32) were examined using ten SYBR® Green qPCR assays to evaluate the reproducibility of the SingleShot™ SYBR® Green Kit. Cq, quantification cycle.

Data used with permission from Gert Van Peer, Pieter Mestdagh, and Jo Vandesompele, Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Optimal Efficiency and Dynamic Range across Expression Ranges

PCR Efficiency

Three targets were analyzed using input cell numbers ranging from 10 to 105 HeLa cells.

PCR efficiencies greater than 98% were maintained regardless of the expression levels, and sensitivity down to 10 cells was observed.

Accurate gene expression studies require that PCR efficiencies are within 90-110% per the MIQE guidelines.

Accurate gene expression studies require that PCR efficiencies be within 90–110% per the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. The SingleShot RNA control was used to monitor PCR inhibition. As the control quantification cycle (Cq) values remained constant, no inhibition was noted.


SingleShot™ SYBR® Green One-Step Kit enables a large dynamic range

Two technical lysate replicates were examined across three differentially expressed genes (B2M, HPRT, TBP) using a human kidney tumor (HKT) cell line.

The SingleShot™ SYBR® Green One-Step Kit showed a large dynamic range across all assayed expression levels.

The SingleShot™ SYBR® Green One-Step Kit showed a large dynamic range across all assayed expression levels

Cq, quantification cycle.

Dynamic Ranges of SingleShot™ One-Step Kit Lysates and Isolated RNA

SingleShot™ SYBR® Green One-Step Kit lysates yield a dynamic range similar to purified RNA

Gene expression data were generated from a 10–105 dilution series of K562 human cell line and compared using SingleShot™ SYBR® Green One-Step Kit lysates and isolated RNA (Aurum™ Total RNA Mini Kit).

B2M expression was assessed using a PrimePCR™ qPCR Assay. The lysates were generated without the need for RNA isolation.

Equal Performance and Superior Sensitivity Compared to Isolated RNA

 

SingleShot™ Probes One-Step Kit lysates yield a dynamic range similar to purified RNA

Gene expression data were generated from a 10–105 dilution series of K562 human cell line and compared using SingleShot Probes One-Step Kit lysates and isolated RNA (Aurum Total RNA Mini Kit).

TBP expression was assessed using a PrimePCR™ qPCR Assay. The lysates were generated without the need for RNA isolation.

Dynamic Range of SingleShot™ Probes One-Step Kit Lysates vs. RNA

RFU, relative fluorescence units.

The SingleShot family of kits provides a complete and fast solution for generation of lysates from cell cultures, optimized for downstream one- or two-step quantitative PCR (qPCR) reactions, without an RNA purification step.

Single Shot Workflow

Kits are available in SYBR® Green or probe chemistries. A cell lysis kit is also offered as a stand-alone option.

All kits offer the following key benefits:

Choose the Right Kit for Your Needs

SingleShot Kit Features Product Options
Cell Lysis Kits
  • Cell lysis for RT-qPCR
  • RT-qPCR reagents sold separately
  • No RNA purification needed
100 reactions
(172-5080)
500 reactions
(172-5081)
Cell Lysis Two-Step RT-qPCR Kits
  • Cell lysis, reverse transcription, and qPCR
  • All two-step RT-qPCR reagents included
  • No RNA purification needed
SYBR® Green
100 reactions
(172-5085)
Probes
100 reactions
(172-5090)
Cell Lysis One-Step RT-qPCR Kits
  • Cell lysis, reverse transcription, and qPCR
  • All one-step RT-qPCR reagents included
  • No RNA purification needed
SYBR® Green
100 reactions
(172-5095)
Probes
100 reactions
(172-5070)
 

Simple Workflow

Simple, short, minimal hands-on protocol is suitable for automation and provides a high-throughput solution.

Grow cells to desired density, transfer up to 105 cells to wells of a PCR plate, add lysis solution, and incubate at room temperature for 10 minutes.

Heat for 5 minutes at 37°C and then for 5 minutes at 75°C. The lysate is ready for RT-qPCR or can be stored for future use.

Preamp Single Shot Workflow

Cell Lines Validated for Use with the SingleShot Cell Lysis RT-qPCR Kits

All SingleShot Cell Lysis RT-qPCR Kits have been validated for use with a number of common human and rodent cell lines, such as all types of adherent and suspension cells, including but not limited to:

Cell Line Property Species
A549 Adherent H. sapiens, lung carcinoma
HeLa Adherent H. sapiens, cervical adenocarcinoma
HepG2   Adherent  H. sapiens, liver carcinoma
NIH/3T3 Adherent M. musculus, embryonic fibroblast
MCF-7 Adherent  H. sapiens, epithelial, adenocarcinoma
NCI-H1975  Adherent H. sapiens, lung adenocarcinoma 
K-562 Suspension H. sapiens, chronic myelogenous leukemia
HEK 293 Suspension H. sapiens, kidney
Jurkat Suspension H. sapiens, T lymphocyte
PC-12  Suspension R. norvegicus, pheochromocytoma, adrenal
HepRG  Primary-like H. sapiens, hepatocytes
Storage at 4°C 6 months:
Lysis buffer

3 months:
iTaq™ Universal One-Step Reaction Mix
SsoAdvanced™ Universal Supermix
Storage at –20°C  All kit components are stable for 12 months
SingleShot™ Cell Lysis Kit
SingleShot™ Cell Lysis Kit

172-5080
Pkg of 1, kit includes 5 ml (1 x 5 ml) of cell lysis buffer, 100 µl of proteinase K solution, and 100 µl of DNase solution, for 100 x 50 µl reactions

List Price:   $450.00
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SingleShot™ Cell Lysis Kit

172-5081
Pkg of 1, kit includes 25 ml (5 x 5 ml) of cell lysis buffer, 500 µl of proteinase K solution, and 500 µl of DNase solution, for 500 x 50 µl reactions

List Price:   $2,000.00
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SingleShot™ SYBR® Green Kit

172-5085
Pkg of 1, kit includes SingleShot Cell Lysis Reagents, iScript™ Advanced cDNA Synthesis Kit for RT-qPCR, SsoAdvanced™ Universal SYBR® Green Supermix, and the SingleShot RNA Control, for 100 x 50 µl reactions

List Price:   $1,100.00
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SingleShot™ Probes Kit

172-5090
Pkg of 1, kit includes SingleShot Cell Lysis Reagents, iScript™ Advanced cDNA Synthesis Kit for RT-qPCR, SsoAdvanced™ Universal Probes Supermix, and the SingleShot RNA Control, for 100 x 50 µl reactions

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SingleShot™ SYBR® Green One-Step Kit

172-5095
Pkg of 1, kit includes SingleShot Cell Lysis Reagents, iTaq™ Universal SYBR® Green One-Step Kit, and the SingleShot RNA Control, for 100 x 50 µl reactions

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SingleShot™ Probes One-Step Kit

172-5070
Pkg of 1, kit includes SingleShot Cell Lysis Reagents, iTaq™ Universal Probes One-Step Kit, and the SingleShot RNA Control, for 100 x 50 µl reactions

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Number Description Options
6604 SingleShot™ Cell Lysis RT-qPCR Kits Application Note, Rev A Click to download
10042473 User Manual, SingleShot™ Probes Kit, Rev A Click to download
10042474 User Manual, SingleShot™ SYBR® Green Kit, Rev A Click to download
10042475 User Manual, SingleShot™ SYBR® Green One-Step Kit, Rev A Click to download
10042476 User Manual, SingleShot™ Probes One-Step Kit, Rev A Click to download
10042477 User Manual, SingleShot™ Cell Lysis Kit, Rev A Click to download
6572 Accurate and Reproducible RT-qPCR Gene Expression Analysis on Cell Culture Lysates Using the SingleShot™ SYBR® Green Kit, Rev A Click to download