Oligo dT and random hexamers are two different primers used in reverse transcription reactions for subsequent real-time PCR for gene expression. I usually use oligo dT primers. But in the lab I stayed now, random hexamers were used by everyone except me. The expression levels of genes varied with these two primers even using an identical template RNA. Why? Oligo dT primers only prime at the poly-A tail of mRNAs, so this is where the reverse transcriptase starts. All cDNAs made this way will contain the 3' end of the gene. If a gene is long, the enzyme can sometimes fall off, so that the 5' end is missing. Random hexamers, though, prime randomly along the RNAs, so that the cDNAs resulting from this method represent bits and pieces of the gene along its entire length, but all regions of the gene may not be equally represented. So all regions of the gene may not be made into cDNA equally by the two methods, oligodt priming has bias towards the 3' end of the transcripts, random priming tend to over-represent the central part of the transcripts. None is perfect. What's your opinion about it?
标签: RT-PCR
