Since the amount of total RNA in the plasma is very low, about 50 ng, I wonder if I can do cDNA synthetise at the same time for my special RNA and housekeeping?
Hi Elahe,
as I can understand you want to amplify two cDNA in the same reaction. it's named multiplex PCR. of course you can, at the critical point that Tm and protocols should be relatively same for both genes. take attention of the relative amount of produced cDNA since pairs of primers won't necessary have the same PCR efficiency, changing the initial proportion of the genes in the original sample.
fred
Theoretically it is possible.
In Addition to Frederic I think : enzyme (Reverse transcriptase) and substrate ( dNTP) And other factors may also be limiting factor . Although this can be solved by decrease the cycle number. if it works well In that case you will get less amount of cDNA in the end of cycle.
Hah? Possibly I understood the question wrong ... You do have RNA and want to synthesize cDNA, fine ... normally you are intersted in genes and their expression. So, mRNA with a A tail should be the template of your cDNA reaction ... use oligo dT primers for the cDNA synthesing reaction. Using this protocol only the subspecies of mRNA is reverse transcribed. This is 3% of total DNA amount (regarding literature knowledge). Thus, you should not have probs with too less enzyme or dNTPs. If your amount of RNA is low, then nearly all mRNA should be a template of one cDNA strand (if there is enough nucleotides). And AFTER that you can amplify your genes of interest in a multiplex PCR.
It's not common to reverse-transcribe only one (or two) gene/s of interest from a RNA sample.
Is there a special need for that?
If a template is too rare in the cDNA of oligo dT based reverse trancription, it is possibly not in your sample ... you can not really deal with this issue by a "sequence specific" reverse transcription, I think. use more material or use any other technique to enrich it.
Hello Jörg,
thank you for your comment, but I work on miRNA not mRNA
- Badges
- Science method