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

Page 8

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10042473

SingleShot

™

Probes Kit

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. A deviation from linearity results
from incomplete lysis and/or RT-qPCR inhibition.

Using the SingleShot RNA Control to Determine Optimal Input Lysate Volume

1. Resuspend the RNA control template in 200 µl of nuclease-free TE buffer pH 7.5.

2. Prepare cell lysate from either adherent (see Processing of Adherent Cells in a

96-Well Culture Plate section) or suspension cells (see Processing of Nonadherent
Cells in a 96-Well PCR Plate section) with an optimal number of input cells.

3. Vary input cell lysate in the reverse transcription reactions as shown in Table 8.

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

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