Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits User Manual

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SingleShot

™

SYBR

Green Kit

Fig. 1. Determining optimal cell input number. In this example, 10

input cells is the maximum input.

No inhibition was noted across the input series. Target genes demonstrate linearity across all four logs.

0 1 2 3 4 5

Cell number, log

Target 1

Target 2

Control RNA

6. Mix the qPCR reaction mix thoroughly to ensure homogeneity and dispense equal

aliquots into each PCR tube or into the wells of a PCR plate. Use good pipetting
technique to ensure assay precision and accuracy.

7. Add cDNA to the PCR tubes or wells containing qPCR reaction mix (prepared using

Table 7), seal tubes or wells with flat caps or optically transparent film, and gently
vortex to ensure thorough mixing of the reaction components. Spin the tubes or plate
to remove any air bubbles and to collect the reaction mixture in the vessel bottom.

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 log of the
number of cells used to generate the lysate (Figure 1). A constant Cq value
across the input cell range indicates complete lysis and no RT-qPCR inhibition.
A deviation of >1 Cq value indicates incomplete lysis and/or 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