Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 7)

© 2014 Bio-Rad Laboratories, Inc. 10042477
SingleShot ™ Cell Lysis Kit
6. Perform data analysis according to the following guidelines:
■ ■
RNA control: Plot the Cq values for the RNA control against the percentage input
lysate (Figures 2A and 2B). A constant Cq value across the input lysate range
indicates no RT-qPCR inhibition. A deviation of >1 Cq value indicates RT-qPCR
inhibition. In the example shown in Figure 2A, optimal performance can be
achieved with 100,000–10 input cells
■ ■
Target gene: If a target gene is amplified in parallel with the RNA control, plot the
Cq values for the target gene against the percentage input lysate (Figures 2A and
2B). A decrease in Cq value is expected as input lysate increases. Deviation in
the linear response of the RNA control results from incomplete lysis and/or from
RT-qPCR inhibition
A
B
Cq
27
26
25
24
23
22
21
20
19
10 20 30 40 45
Input lysate, %
Target
Control RNA
Fig. 2. Determining optimal input cell lysate amounts. A, the maximum allowable cell lysate input
is 45%. B, this one-step RT-qPCR reaction shows no PCR inhibition across all tested input lysate amounts.
Cq, quantification cycle.
Cq
30
20
10
0
10 15 20
Input lysate, %
Target
Control RNA