Dear All

I was wondering what the difference was between using oligo-dT primers and random primers when reverse transcribing samples of RNA into cDNA for use in PCR? How does their use effect the code, length etc. of the cDNA. What other primers could be used?

Thanks very much for the help

Phago

Hi Phago,

Basically there are three types of primers used in RT. They are oligo dT, random primer and gene specific primer. Oligo dT primers only bind to poly A tail of mRNA and thus has good specificity. Its diaadvantage is if an mRNA is too long, the reverse transcription may not be able to reach the 5' end. But if primers for RT-PCR are designed close to the 3- end of the mRNA, this should not be a problem.

Random primer can bind to any RNA such as mRNA and rRNA, the latter contibutes to 95% of total cellular RNA, and results in cDNA with greater complexity than oligo dT. Random primers can be used if mRNAs don't have or have short poly A tail, RNA sample is mRNA, etc.

cDNA synthesized with a gene specific primer has the best specificity but can only be used to amplify that specific gene in subsequent RT-PCR.

Hope that helps.

Green