cDNA quantification for PCR - (Feb/27/2008 )
I have been optimising my RT-PCR protocol for analysis of rat brain samples and have become rather confused about whether or not I should quantify my cDNA. Following RNA extraction I calculate RNA concentration using the A260 and purity with A260/A280 and then add 2ug RNA to each RT reaction. The protocol commonly used in my lab then says to calculate the concentration of cDNA following reverse transcription using A260 to then add 2ug of cDNA to each PCR reaction. Now this is where I get confused because the A260 readings are all very similar even in the RT negative samples, I assume this is because they all contain the same amount of dNTPs. If this is the case, is there any point in doing this step at all or should I just add the same volume of RT product to each PCR reaction (as many other protocols seem to suggest)?
I hope someone can help me out here as people in my lab seem to be as confused as me!
you can try to quantify your cDNA but then you have to purify it first to get rid of the dNTPs and enzymes,....but i don't think that a spectrophotometer is sensitive enough.
but in my opinion it is not necessary. usually you put in your 20µl RT reaction for example 2µg RNA. after the RT you assume that you have 2µg cDNA (if you used random primers) that means, you have 100ng / µl cDNA. for your PCR you use about 100 ng (= 1µl) as template. you can also dilute it with water so that you don't have to pipet 1µl e.g. to 50 ng or 25 ng / µl.
if you use oligodT primers, then simply use 1/20 of your RT reaction for PCR.
For quantification, I always purify (gel extract) my samples before I determine their concentrations.
I don't usually quantify it. Won't an rt-pcr with a housekeeping gene be enough? Ok, if you want to be very precise, 2 housekeeping genes
when doing rt-pcr for some endosperm samples i used 2 hkg's (actin and ubiquitin) and there were huge differences. i realized a critical part was having the same amount of total RNA when synthezising cDNA
Thanks for your advice folks!
So as I suspected taking the A260 readings of the RT product is a waste of time? I am already using a housekeeping gene so I will just continue using equal amounts of RNA in my RT and then equal volumes of the RT product in my PCR and hopefully all will be well!