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?.
I would concur with the above but also add that primers and in particular exon spanning primers that just target mRNA and thus circumvent problems associated with extraneous signal linked to genomic DNA should be biased towards the 3 prime end of the coding sequence. Specifically and as alluded to above if you make cDNA by reverse transcription using oligo dT which primes the poly A tail of mRNA you should situate your primers over contiguous exons 1-2 in from the poly A tail including intact the 3' UTR. This is because the efficiency of RT dewhereclines declines with oligo dT 2kb upstream from the poly A tail and thus efficiency of downstream PCR is reduced. In contrast if you perform RT with random hexameter it is usually possible to site primers any where along the message including the 5' exon
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
I would concur with the above but also add that primers and in particular exon spanning primers that just target mRNA and thus circumvent problems associated with extraneous signal linked to genomic DNA should be biased towards the 3 prime end of the coding sequence. Specifically and as alluded to above if you make cDNA by reverse transcription using oligo dT which primes the poly A tail of mRNA you should situate your primers over contiguous exons 1-2 in from the poly A tail including intact the 3' UTR. This is because the efficiency of RT dewhereclines declines with oligo dT 2kb upstream from the poly A tail and thus efficiency of downstream PCR is reduced. In contrast if you perform RT with random hexameter it is usually possible to site primers any where along the message including the 5' exon
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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