I have cDNA samples extracted from 90+ individuals which I intend to utilise in a series of qPCR experiments to assess the influence of a specific SNP genotype/s on target gene/s expression. I was unaware at the time that no-RT controls were needed, and it is only now as I prepare to perform qPCR that I have realised that this is recommended.
I am able to synthesise these controls, however was hoping whether someone could clarify:
(1) if a no-RT control is recommended for each cDNA sample prepared?, and if so
(2) whether a no-RT control is recommended for every sample in the qPCR run (i.e., three technical replicates + no-RT control)? I'm assuming no-RT controls would not be required in replicates?
Other QC parameters have been applied: All RNA samples were purified prior to cDNA synthesis, and NTCs were included in every batch. Primers have been designed across two exons, etc.
Many thanks in advance!
Hi Kealan,
in principle, if a no-RT control is required it has to be done for every RNA sample and PCR target. However, if your target gene allows a proper primer design targeting cDNA from spliced RNA only and no pseudogenes are present I would recommend to test your primer on cDNA and noRT, gDNA controls of some individuals. If you get the specific PCR product only with the cDNA samples but not the controls I would suggest that you don´t need to run no-RT controls for all of your samples. If you are not lucky and your primers amplify DNA you should do no-RT controls. However, I would suggest that you can put RNA equivalent to the amount of cDNA per PCR reation directly into the qPCR to check for contaminating DNA.
Best,
Norman
One way to simplify the amount of controls is to run -RT for your wild-type (or your baseline control), for a sample would have the "normal" amount of the gene present.
Really it's to check for contaminating gDNA, which would be an undesired outcome of your RNA extraction and cDNA synthesis. You can DNase your cDNAs as a way to minimize gDNA carryover.
In short, you need it for at least 1 sample, but should not need it for all samples unless you get significant gDNA contamination (in which case Dnase is recommended)
Norman Hafner Amy Klocko Thank you very much for your suggestions!
All RNA samples have been treated with DNase as part of the purification procedure (this is part of our lab's standardised RNA extraction process).
I think I may apply a combination of both approaches suggested - synthesise no-RT 'cDNA' for the baseline controls to test the primers prior to qPCR and include these as part of the qPCR as an additional sanity check.
Your input is greatly appreciated!
Kealan
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