first, i am new to rt-qpcr.
i am trying to estimate several gene expression in yeast using rt-qpcr.
i am thinking about using random primer for cdna synthesis with invitrogen vilo kit (it has a random primer mixture).
in the process of learning more about random primer, i encountered a problem and got quite puzzled:
random primer will produce shorter fragment of cdna compared to oligo(dt), and the average cdna length is dependent on random primer:rna ratio.
for qpcr, we need a set of specific primers to amplify some regions of the interested genes (i think it is recommended amplified region <150bp).
so, i am thinking: when we keep increasing random primer:rna ratio in the rt step, the obtained cdna should also get shorter and eventually below 150 bp; when it is so short, it can not be amplified by pcr primers any more (like cutting a dna in the middle of the pcr amplification region), this should lead to higher apparent ct and loss of linearity between ct and amount of added rna.
however, in invitrogen introduction, this kit has a linear response from 1pg to 5ug rna, over 1E6 fold change.
maybe something wrong with my understanding:
1) with increasing random primer:rna ratio, is there a limit for the synthesized cdna? or it can be very short?
2) for a single rna molecule, can it bind multiple primers and produce multiple cdna fragments at the same time?
any input will be greatly appreciated.
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question about using random primer for rt-qpcr
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