I am designing primer for real time pcr but i have some confusion in selecting the sequence. Is it the mRNA sequence or CDS of respective gene? or we can select anyone?.
In principle, you can measure transcript levels by qPCR using oligos that recognize either the coding sequence or UTRs, although the majority focus on coding sequence. Oligos that span exons have the advantage that they are usually selective for cDNA over genomic DNA (provided the intron is large enough, several hundred bp or more).
Use CDS sequence. You can use OligodT or Random Haxamer for the universal.
If you are asking about complementary DNA sequence, then it depends on the RT step. If you are only performing first strand synthesis with random hexamers or oligo dT, then the resulting product will all be complementary sequence. If you perform second strand synthesis, then the products are double stranded cDNA, and then you don't need to worry about whether your qPCR oligos anneal to the coding or non-coding strand.
Got it, thanks to all of you for kind suggestions and informations
Daniel M Cohen, Maulik Rachh, M N Manoj
Hi, Complete coding sequence (CDS) is best for both qPCR and RT-PCR because cds does not contains intron it contains only exons but mRNA sequences contain both coding and non-coding sequences..
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