Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 6)

© 2014 Bio-Rad Laboratories, Inc. 10042473
SingleShot ™ Probes Kit
6. Load the PCR tubes or plate into the real-time PCR instrument and start the
PCR run.
7. Perform data analysis according to the instrument-specific instructions.
Recommendations for Assay Design and Optimization
■ ■
For best qPCR efficiency, design assays targeting an amplicon size of 70–150 bp
■ ■
The SsoAdvanced Universal Probes Supermix and the recommended qPCR
cycling protocols have been optimized for assays with a primer melting
temperature (T
m
) of 60°C designed using the open source Primer3, Primer3Plus,
or Primer-BLAST programs at their default settings. If primers are designed using
other programs, adjust the annealing temperature accordingly
■ ■
The probe’s T
m
should be 8–10°C higher than the calculated primer T
m
■ ■
In a duplex reaction, for best results apply the brighter fluorophores to the lower-
expressing targets and the dimmer fluorophores to the higher-expressing targets
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 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.
Table 6. Preparation of SingleShot cell lysis master mix containing the RNA control.
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
RNA Control Template 5
Total volume 250