I need to design a set of primers that will be used for Reverse Transcriptase PCR, and Real Time PCR. I want to use the same primer set for both procedures. Should I use the mRNA sequence as Reference Sequence or genomic DNA when I'm designing the primers? Should I use RNA primer or DNA primer?
Think out the terminology you used here, Reverse transcript: mRNA to cDNA. Your RT or qRT PCR template will be cDNA (not mRNA). In this case if you used primer set designed from mRNA, what will be the fate of U (uracil)? So, in most of the case so far we design primer sets from the gDNA. To design primers with probe, or without probe for qPCR or others, you can used the idt or primer3.
I believe we should design primers from the cDNA sequence (i mean to say functional gene without introns), not mRNA, not genomic DNA. Not choosing genomic DNA is importnat especially when we don't do DNase treatment for RNA samples. If we design using genomic DNA, what if you have genomic DNA contamination in RNA samples. The DNA sequence for a beta-actin gene can be found in CCDS of https://www.ncbi.nlm.nih.gov/gene/?term=beta+actin page (example). You can verify whether your gene set codes for functional gene or genome at https://genome.ucsc.edu/cgi-bin/hgPcr.
it doesn't matter which one you use. The primers you're designing are expected to work both with mRNA and gDNA, so the primers need to anneal to both templates. The templates are different but the target sequences are the same.
More generally, if C needs to match A and B, then the matching area is identical between A and B. It works with both.
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