cDNA before qPCR-purifying or not?! - (Jun/11/2005 )
I am very confused about this one. I have faced the question whether i should purify my RT sample or not before running in qPCR. In one-step RT-qPCR then we run alltogether in one tube without purifying cDNA. I wonder should i purify my RT sample before running in PCR in a two-step Rt-qPCR? Should i sequence my cDNA before running it in qPCR?
Thanks for any ideas!
Are you you a gene specific primer for generating the transcript of the RNA? If not, you cant sequence it. Well, you can sequence it, but you will get a scrambled read... in addition to which, unless it is a massive rt rxn, I really doubt you will get enough cDNA to get decent sequence.
Secondly, how would you purify it? I personally don't know how to purify it after the rxn... I suppose you could use a quiagen kit thing.. but why? Why do you think you need to purify?
If you are concerned about background amplification from the RNA and genomic dna in the rt rxn, just set up a negative control (rt rxn with every thing but the reverse transcriptase) and run it side by side with your unknown on the real time machine to see what your background amplification is.
I see people talk about purifying and sequencing their cDNA and i wonder if this only happens in other applications and not in real time qPCR?
Hope for any inputs!