Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 26)

20 | SsoAdvanced ™ Universal Probes Supermix Instruction Manual 20 |
If your no-RT control wells indicate amplification, you need to determine the amount
of gDNA contamination present in your cDNA sample(s) to understand the impact on
your data.
1. Using Table 5, determine the percent of gDNA contamination present. For example, if the
∆Cq (no-RT control Cq – cDNA Cq) for a given sample is seven or greater, then less than 1%
of the DNA present in the sample is gDNA, which would be considered insignificant.
Table 5. Determining percent of gDNA contamination.
∆Cq
Percent Contribution, %
1 50.00
2 25.00
3 12.50
4 6.25
5 3.125
6 1.5625
7 0.78125
2. Evaluate the assay design and note the location of the primers. To avoid gDNA amplification,
at least one primer must span an exon:exon junction site. Alternatively, the primers can be
designed in two different exons that are separated by an intronic region >1 kb.
If you are using an internal positive control (IPC) and the standard deviation of the Cq values
across all samples is >0.167, then consider the following:
When the IPC for a given sample(s) is higher than the group, this is most likely due to the
presence of a PCR inhibitor. Review the sections on sample preparation for more information.
Multiplexing
Ideally, data between singleplex and multiplex should remain the same in terms of Cq values
and PCR efficiency. In addition, if your data exhibits relative Cq shifts for all data points
between singleplex and multiplex, then the final data output remains the same. However, if
you observe variable Cq shifts for respective data points between singleplex and multiplex,
consider the following:
The higher expressing assay may be using up the reaction components such as dNTPs
and enzyme, and thus causing a shift in the lower expressing assay to later Cq values than
observed in singleplex.
1. Primers and probes from different assays may be interacting. Make sure there are no stable
dimers formed between the oligos from different assays. This can be completed using
various open source tools online.