Something about absolute quantification... - Please help me understand this!! (Oct/23/2007 )
I was reading about absolute quantification method... I cannot fully understand the phrase:
"Generally, it is not possible to use DNA as a standard for absolute quantitation of RNA because there is no control for the efficiency of the RT-step"
Im performing abs.quant. to determine most stably expressed housekeeping genes in a group of samples... so I compare expression ratios of these genes using cDNA (not working with any target gene for now), but the standard curve I generate is from genomic DNA!! And if I read the quoted phrase, this is wrong!!
Is this really and terrifyingly wrong? I dont understand why if I use cDNA instead of genomic DNA for the standard curve, I would have an "efficiency control of the RT-step". Can anybody explain me that?
I hope I made myself clear...
Thanks for helping me with this mess on my head!!
I think it means that the perfect way to do it would be to have a known amount of RNA, and RT a standard curve prepared from that together with your samples (same buffer, same conditions, same mistakes). RT may work better/worse on more/less RNA, so it may distort your standard curve a little.