Hi everyone, just a quick question.
I am using a library protocol that uses a magnetic poly(A) isolation of mRNA, followed by a random hexamer based first strand cDNA synthesis step. The poly(A) kit is expensive and doesn't work well on smaller sample sizes, which I am using. My question is, why don't I just use a oligo(d)T based primer instead of a hexamer primer in my first strand synthesis and skip the poly(A) magnetic isolation. Why doesn't everyone do this instead of a magnetic poly(A) isolation step... do I expect more yield losses or rRNA contamination, or a substantially greater bias towards the poly(A) tail?
Thanks in advance,
Mike
I am using a library protocol that uses a magnetic poly(A) isolation of mRNA, followed by a random hexamer based first strand cDNA synthesis step. The poly(A) kit is expensive and doesn't work well on smaller sample sizes, which I am using. My question is, why don't I just use a oligo(d)T based primer instead of a hexamer primer in my first strand synthesis and skip the poly(A) magnetic isolation. Why doesn't everyone do this instead of a magnetic poly(A) isolation step... do I expect more yield losses or rRNA contamination, or a substantially greater bias towards the poly(A) tail?
Thanks in advance,
Mike
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