I wonder if the positive control primers and probes authorized by the US CDC for amplifying human RNAse P are specific to the RNAse P chromosomal DNA. My thought process is that if primers are created that are specific to cDNA for RNAse P (i.e. the primer crosses a region between two exons, eliminating it's ability to bind to the chormosomal gene) then those primers could be used to validate the reverse transcription step in RT-PCR.
#covid19 #RT-qPCR #diagnostics
Dr NICO: Please take a look at the following links:
https://www.researchgate.net/post/Is-anyone-concerned-with-CDC-Covid-19-kits-RNAse-P-controls
https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf
it appears that RNAse P is all one exon, which to me makes it seem like a poor candidate for discriminating between gDNA and cDNA products from a PCR reaction...
This has led me to the idea of designing primers that amplify cDNA, but not gDNA in order to control for the the reverse transcription step in diagnostic RT-PCR including those for detecting COVID. I found this paper that outlines a list of human housekeeping genes (that have similar expression levels across cell types) and that have similar isoforms expressed across cell types (i.e. the mature mRNA transcripts are made up of the same exons)
My new idea is to design PCR primers that will only anneal to the cDNA template, and not gDNA template. The way that I would do this is to design primers that anneal to a region that includes an exon junction, and empirically test whether those primers work to successfully discriminate between cDNA and gDNA of those human genes.
Does this seem like a good approach?
Why or why not?
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