Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 8)

© 2014 Bio-Rad Laboratories, Inc. 10042474
SingleShot ™ SYBR ® Green Kit
Table 6. Preparation of SingleShot cell lysis master mix for optimizing input cell number.
Reagent
Reagent per 4-log Tenfold Dilution Series
in a 96-Well Plate, µl
SingleShot Cell Lysis Buffer 235
Proteinase K Solution 5
DNase Solution 5
SingleShot RNA Control Template 5
Total volume 250
* Scale all components proportionally according to sample number and reaction volumes.
Table 7. Preparation of qPCR reaction mix for RNA control assay.*
Component
Volume per 10 µl
Reaction, µl
Volume per 20 µl
Reactions, µl
Final
Concentration
SsoAdvanced ™ Universal SYBR ®
Green Supermix (2x)
5 10 1x
SingleShot ™ SYBR ® Green
qPCR Control Assay
0.5 1 1x
cDNA (add at step 8) 1–2 2–4 —
Nuclease-Free Water Variable Variable —
Total reaction mix volume 10 20 —
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
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.