To perform a two-step RT-qPCR (starting material is RNA, then reversed transcribed to cDNA followed by cDNA amplification), do we have to design the sequence-specific primers for cDNA amplification in the 2nd step with reference to the mRNA sequence or the cDNA sequence?
Conversely, in the one-step method, as the primers should be gene-specific, should they be designed with reference to the exons in the mRNA sequence. Can anybody please help me with these two questions??
1. For your purpose, the mRNA and cDNA sequences are equivalent. Why do you believe there would be differences affecting your primer design process? Physically, the DNA component is of course what's amplified. Primers that pair with the original mRNA strand aren't productive, and you don't need to think about that. In the 2nd PCR cycle, they'll have DNA template.
2. Yes, you must target the exons, because there are no introns in your cDNA. If you want to make a primer that works only on cDNA, and does not amplify genomic DNA, you can bridge a long intron with your primers. This is helpful for eliminating false signal from contaminating genomic DNA. PrimerBlast is a great way to do all this automatically.
I recommend you to this page https://eurofinsgenomics.eu/en/dna-rna-oligonucleotides/oligo-tools/qpcr-assay-design-tools/
Hope it will help answer your worries.
Good luck
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