Are primers used in reverse transcriptase PCR also used in real time PCR?
Or should I redesign the appropriate primers? If so, what is the difference?
In reverse transcription, usually oligo dT primers are used which hybridizes to the polyA sequence on mRNA.
For real-time, you should have primers specific to the gene of your interest.
Hi!
It depends on the method that you use: One-step or Two-step assay.
One-step assays combine reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. So after RT-PCR (cDNA synthesis), it will directly continued with qPCR. One-step RT-qPCR only utilizes sequence-specific primers, so that you will use the same RT-qPCR primers.
In two-step assays, the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies. If you use two-step assays, you can choose three different approaches for priming cDNA reactions in two-step assays: oligo(dT) primers, random primers, or sequence specific primers (qPCR primers). However, if you use the qPCR specific primer in the cDNA synthesis, the synthesis is limited to one gene of interest .
Thank you,
Best regards
You need to design a primer of your gene of interest whether it's one step or two steps assay. Gene of interest should be mRNA.
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