Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 5)

© 2014 Bio-Rad Laboratories, Inc. 10042477
SingleShot ™ Cell Lysis Kit
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. Follow the previously described RT-qPCR reaction
setup and include:
■ ■
For one-step and two-step RT-qPCR, use 1 µl of the PrimePCR Reverse
Transcription Control Assay (SYBR
® Green or probe, respectively) per 20 µl
RT reaction
■ ■
If desired, gene expression targets of interest can be amplified in parallel with the
RNA control assay in singleplex reactions when using SYBR
® Green chemistry.
For probe-based chemistry, the HEX-labeled control assay may be duplexed with
a non-HEX labeled probe for a gene expression target.
5. Perform data analysis according to the following guidelines:
■ ■
RNA control: Plot the Cq values for the RNA control against the log input cell
numbers used to generate the lysate (Figure 1). A constant Cq value across
the input cell range indicates no RT-qPCR inhibition. A deviation of >1 Cq value
indicates RT-qPCR inhibition. Input cell numbers that show such a Cq deviation
should be avoided. In the example shown in Figure 1, optimal performance can
be achieved with 100,000–10 input cells
■ ■
Target gene: Plot the Cq values for the target gene against the log of the number
of cells used to generate the lysate (Figure 1). A decrease in Cq value is expected
as cell number increases. The decrease in Cq values should be linear for cell
numbers that don’t exhibit inhibitory effects. Deviation from linearity results from
incomplete lysis and/or RT-qPCR inhibition
Table 5. 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