Eon's mommy, on Dec 10 2009, 08:44 AM, said:
I have one BIG problem with my GOI and their dilutions. I'm trying to do a validation experiment for DDCt method (The comparative Ct method) with SYBR Green. I've used 1 ug of total RNA for reverse transcription and after that I did five points serial dilution (1:5, 1:10, 1:50, 1:100 and 1:500) with my cDNA. Housekeeping gene is working well (efficiency=95,7%, RSq=0,999) but my GOI doesn't work at all. Well if I am precise I've got three Ct's, two within 1:5 dilution (32.95 and 33.73) and one in 1:50 dilution (33.79)
. It is very strange to have so little differences between Ct's over ten-fold dilution, is it not? What am I doing wrong? Dissociation curve is ok, just one peak for reference gene and GOI. I've put all results in an attachment.
Final volume of qPCR reaction was 20 ul. For GOI I've used 300 nM primers and 100 nM for housekeeping gene.
1 ul of sample was used per reaction.
I don't know what went wrong
. Could you help me out? What are my solutions?
P.S. One stupid question for the end. Can someone do primer optimization on genomic DNA and then use it on cDNA samples or not? And if not, why?
When I've done validation curves, I've always done them over 4-6 LOG dilutions to get a good range. However, based on your Ct values for a 1:5, my guess is that you just don't have that much transcription occurring in whatever sample you've got. Most likely, the similarity in Ct between the 1:5 and 1:50 is because there's just too little too measure. Are you running NTCs along with these, or -RT controls to make sure it's not low level genomic DNA giving you the signals? Alternatively, check the melt curve analysis of your products. I had a set of primers once that amplified well and gave a Tm of about 80C, but in -RT samples there were primer dimers that game a peak at about 75C, and when I did -RT LOG dilutions I got a flat line, because every sample had the same Ct due to primer dimers, and it was about 30-35 at each point. The thing is, the melt curve for -RT and +RT both gave only one peak, but Tm of the peaks was different.
If you have a reaction where you know the GOI amplified (maybe from gDNA), make sure the Tm of that product matches the Tm of the product you're getting in your validation curve. If that's not the problem, then perhaps it's just too little mRNA present to give a sufficient amount of cDNA for a good curve. A colleague of mine is having that trouble right now with some bacterial GOIs. He has to really concentrate the mRNA to get sufficient cDNA in the RT reaction to be able to have a good enough dynamic range for the validation curve. The problem that arises with concentrating the samples that much is that you likely may have a lot of residual gDNA being added, which causes problems with -RT controls and all that.
As for the P.S., my understanding for primer optimization and validation curve is that it's a measure of the primers' ability to amplify DNA. Whether that's gDNA or cDNA I don't think matters. If someone knows of why that would be incorrect, I would like to know as well.