Hello,
We have always used random hexamers in my lab when creating single-strand cDNA for qPCR applications. My understanding is that these primers bind to the RNA and reverse transcriptase transcribes, but due to multiple hexamers binding to the same transcript, you end up with a cDNA pool that is rich with fragmented cDNA. Because a cDNA fragment obviously needs to be the full length of the target region for a set of two primers, I am confused as to how qPCR could give you accurate quantification. Some of the transcripts (perhaps even the vast majority) would be transcribed in such a way that the cDNA would not contain this primer-targeted region. Can anyone offer some insights?
My theory is that this is just a matter of statistics and that some transcripts will inevitably lead to some primer-target-region containing cDNA, but this still leaves a lot of questions.
Real time qPCR utilizes primer pairs that generally give a very short product in the range of 100-150 bp. Random hexamers will usually cover most of the mRNAs so there will be plenty of reverse transcribed cDNAs that span the primer pair target since it is so short. Random hexamers have been used to make cDNA libraries from the very beginning even before PCR because poly DT primed cDNAs are very biased towards the 3' end which is often long and non-coding.
Gregory Dressler Thanks for the insight. When you say there will be plenty of cDNA that spans the primer pair target, there will inevitably be some that does not as well, correct (i.e., perhaps a hexamer binds right in the middle of the target region)? Does normalization help account for this?
Normalization for qRT-PCR usually means normalizing to some internal standard mRNA like actin or tubulin or Gapdh to account for differences in input RNA, but does not account for length of message or the position of the target sequence. If I get what you are saying, you are correct in that the position of the primers could affect the probability of an amplified product, but that probablity would be the same for any two samples being compared.
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