real time RT-PCR problems - (Nov/22/2011 )
I used 140ng/rxn and 150 ng/rxn in two different rxns and I got ~700ng of cDNA from each rxn. I used 100 ng of cDNA/rxn for qPCR. I quantified my samples by Nanodrop. However, all my qPCR failed. Just my +control (home made testis cDNA) worked.
how did you come to your cDNA concentration values? usually, you are not measuring the cDNA conc. after RT because for this you would have to somehow purify the cDNA. In the RT mix are also the primers, dNTPs and enzyme which are interfering with nucleic acid conc. measurement.
Are you sure that your GOI are expressed in your samples? have you tried to measure some housekeeping genes (HKG) in your cDNA which must be present?
Next possibility is that you have inhibitors of PCR in your cDNA. Try to analyze a highly expressed HKG like GAPDH or ACTB in your cDNA with 1) your intended input amount of cDNA 2) with a 1:100 dilution of your cDNA. If only 2) gives a signal you have inhibitors in your RNA/cDNA.
Thank you very much for your reply.
Well, all my quantifications were performed on Nanodrop (RNA and cDNA). It is not the best option, but it is all I have, since I am working with mRNA.
Yes, my target genes are expressed on my samples, and I am already using ACTB primers for all my rxns, but not even ACTB is working with my templates. This is why I am so desperate...
I also runned qPCR using my cDNA (diluted 1:14) and not diluted cDNA, but none amplified.
Do you think it worth repeat the procedure using my cDNA 1:100 diluted?
It can be inhibition of my rxns, but if I have inhibitors on my RNA samples, could they be converted in cDNA?
Thanks a lot for your help. You gave a lot of material to think about it