Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 11)

© 2014 Bio-Rad Laboratories, Inc. 10042474
SingleShot ™ SYBR ® Green Kit
Fig. 2. Determining optimal input cell lysate amounts. The maximum allowable cell lysate input is 45%.
Cq
27
26
25
24
23
22
21
20
19
10 20 30 40 45
Input lysate, %
Target
Control RNA
8. Program the thermal cycling protocol on a real-time PCR instrument according
to Table 5.
9. Perform data analysis according to the following guidelines:
■ ■
RNA control: Plot the Cq values for the RNA control against the percentage input
lysate (Figure 2). A constant Cq value across the input lysate range indicates no
RT-qPCR inhibition. A deviation of >1 Cq value indicates RT-qPCR inhibition.
Input lysate amounts that show such a Cq deviation should be avoided. In the
example shown in Figure 2, optimal performance can be achieved with input
lysate amounts up to 45%.
■ ■
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 (Figure 2).
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
RT-qPCR inhibition.