Is extraction control also add to the qPCR mixture with the sample or does it run separately?
Can anyone please explain their importance and how to use them in q PCR protocol?
Thank you.!
It depends on the pcr mix you are going to use, for example, the ideal is that the extraction control is part of the mix, containing the primers of the species you are making the qPCR. So, you would get the control curve and the target curve. An extraction control performed separately from the sample can be performed, however, the extraction can vary from that performed in the control and in the other samples, so it becomes less reliable. It is very important to have this control, because in a qPCR or rRT-PCR mix where there is no standard internal control cuvette, you can confuse negative results with poorly extracted samples, or with the presence of amplification inhibitors. Read the preprint my department has published (it has been accepted in a peer-reviewed journal and is due to be published in the next few days). He talks a little about amplification inhibitors and methodology to get around this situation in the case of rapid extraction for covid-19. Hope this helps!
False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors
Link: https://www.preprints.org/manuscript/202009.0450/v2
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