Overview
To generate first strand cDNA from mRNA, three priming methods are routinely used, depending on the quality of the starting RNA:
* Oligo (dT)-based priming is preferred when the RNA is of high quality, since first strand polymerization should be continuous from the 3' end of the transcript to the desired sequence regardless of the length of the 3' UTR.
* Random hexamer priming is recommended for fragmented mRNA (lengths <500 bases) where there may be discontinuity between the desired sequence and the poly A tail.
* A primer that is specific to the gene of interest is best when amplifying low abundance transcripts.
Details
Details of this protocol, Gene-Specific RT-PCR, are located on a web site other than Biocompare Protocols.