Which primer concentration for RT with very low RNA amounts? - (Oct/20/2006 )
I have recently started with real time PCR. However, I can only use very low RNA amounts (35ng) per reverse transcription and I have used Sensiscript from Qiagen. My Gene of interest encodes several different variants of 5'end transcripts. First, I used oligo dT primers (10µM per RT-PCR) and the real time PCR worked quite well (even for 1:10 dilution of the RT-PCR products) as long as I amplify segments in the middle of the cDNA of my gene. But I also have to perform real time PCR, where I have to detect the amounts of the different 5'end transcripts. As the Ct values came very late 30 to >35, I thought that the 5'ends of the transcripts are under-represented when using oligo dT primers. Thus, I want to use random-hexamer-primers. Does anyone know which random-primer concentration for reverse transcription is optimal for such low RNA amounts? My problem is, that I have not enough RNA to test different primer concentrations.
Your advice is very much appreciated!
I use Invitrogen's first strand kit, and according to the instructions 50 ng of random hexamers are recommended for up to 5 ug of total RNA. It says "increasing the hexamers to 250 ng per 5 ug RNA may increase the yield of relatively small PCR products (>500 bp), but may decrease the yield of longer PCR products and full length transcripts".
I also use a very low amount of RNA (I don't even try to quantitate it) and get good results with 50 ng of random hexamers. So I would suggest just using the recommended concentration of random hexamers for your kit, especially since you already got good results with oligo dT.