cDNA synthesis using random hexamer vs. oligo dt primers - (Oct/08/2007 )
We are running a real time PCR experiment to study and compare the expression of mRNA in different treatment groups. We have been running our reverse transcription using random hex on the recommendation of a neighboring lab. We then used that synthesized cDNA in our real time PCR runs. While comparison of total RNA is valuable for our study, we are more interested in mRNA expression at the moment. Should we be running our reverse transcription using oligo dt? What is confusing me is that in the literature we used as a guideline for our work, they claim to be studying mRNA expression, while running the reverse transcription using random hexs. Any input would be helpful, thanks for the help.
Each has their own advantages and disadvantages.
Random primers give higher cDNA yield than oligo dT primers, so if your starting RNA is limited, use random primers. OligodT primers offers better specificity because they only anneal to polyA mRNAs. One potential problem with oligo(dT) priemrs is that the reverse transcription may not reach the far 5' end if a mRNA is very long. For this reason I always design my RT-PCR primers close to 3' end of mRNA. I prefer oligodT primers to random primers.
here are some other threads on this question
Just to complicate matters, some report better cDNA production from random 15mers than from random hexamers. I use random 15mers but have never done a parallel comparison with random hexamers, so I can't confirm the claim.