Total RNA in RT-qPCR - (Apr/16/2005 )
Is that only mRNA that we can reverse transcribe into cDNA or can we make cDNA from rRNA and tRNA too? Why i ask this is because some protocols use total RNA in their Reverse Transcription PCR. If mRNA is the only source for cDNA then why don't they extract mRNA from total RNA and then run them to be more precisely?
Thanks for any inputs!
It depends on what kind of primers you are using for reverse transcribing the total RNA. Oligo dt transcribes to yield mRNA, while random hexamers give you generally a mixture. It's assumed that you get the same amount of cDNA sythesized from x amounts of total RNA input used. I don't see any real benefit of isolating mRNA if you are going to quantify using target specific primers. Just my thoughts...
Thanks. Does cDNA mean the product of mRNA or also the product of rRNA and tRNA? Why should anyone use random hexamer since it will give a mixed of products which will contaminate the reaction more easily?
I suppose that if you start even with anchored oligo dT and total RNA, there is not any guaranty that you would have cDNA only from mRNA. Based on my experience, if you start with more isolated mRNA, so you will have cDNA just from mRNA but depend on increasing amount of rRNA in your mRNA you will have a few rDNA in your cDNA. If you check on agoarose gel, you can see ribozomal RNA band.
Also there is huge different intensity for this bands if you use ANCORED oligo dT or ligo dT.