Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 4)

© 2014 Bio-Rad Laboratories, Inc. 10042477
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.
Optimizing Input Cell Number and Input Lysate Amount
For best results, the PrimePCR Reverse Transcription Control Assay, which is sold
separately, can be used to determine optimal input cell number and optimal input
lysate volume.
The PrimePCR Reverse Transcription Control Assay includes a synthetic RNA template
that has no homology to any known sequence and a qPCR assay (SYBR
® Green or
probe) specific for this RNA template.
The template RNA is shipped lyophilized. Upon resuspension, store at –80°C.
Using the PrimePCR Reverse Transcription 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 5.
Table 4. Input volume of SingleShot cell lysate for RT-qPCR reactions.
Two-Step RT-qPCR Input Recommended Input Maximum
iScript Advanced cDNA Synthesis Kit for RT-qPCR
(20 μl reactions)
4 µl lysate 9 µl lysate
SsoAdvanced ™ Universal SYBR ® Green Supermix or
SsoAdvanced Universal Probes Supermix
(20 μl reactions) 2 µl cDNA 4 µl cDNA
Two-Step RT-qPCR Input Recommended Input Maximum
iTaq ™ Universal SYBR ® Green One-Step Kit or iTaq
Universal Probes One-Step Kit (20 μl reactions)
2 µl lysate 4 µl lysate