So I am trying to design the qPCR primers for the mice Sp7. Mice Sp7 has 2 transcript variants, T1, T2, both of them have 2 exon. The T1 T2 only share the same sequence of the exon1/2, while they have 2 different exon2/2 sequence. The problem is, if I have to design a pair of primers for the Sp7, I have to use the exon1 sequence only, which will break the rule of the 2 primers on 2 exons. So what should I do? Can I design 3 primers and use them in the single qPCR reaction well?
You don't always have to design primers to be on different exons. While preferable, it is not always possible and your project illustrates why. As long as you include the gDNA, no RT control, and water control to make sure you are looking at cDNA and not gDNA, you can use primers that are in one exon.
You can't measure the efficiency of qPCR with 3 primers and 2 products. Design two sets of primers: one for variant T1 and a second set for variant T2. Run these in two separate qPCR reactions. Good luck!
Dear
I agree with what mentioned by dear Katie A Clark. It is preferable to use one exon with two primers. I think that . Good luck
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