No-RT control in qRT-PCR - (Feb/28/2007 )
Hope anyone cn help me with the problem occured in my qRT-PCR experiment.
There is an unexpected amplification of the no RT control of my samples. What makes it worse is that the peak in the disssociation curve is the same with the positive cDNA samples. But the ct values for both cDNA samples and noRT samples differ not less than 5 cycles from each other. Is this result valid, or is it really bad?I need to know if there's any acceptable rules in validating the result. I'm so confused and dont know how to prevent gDNA contamination, because i've used gDNA wipeout buffer (quantitech reverse transcription kit from qiagen), and supposely by right it eliminates any possible gDNA contamination.
Another thing, about primer dimer formation in NTC sample. As long as the Tm for both the unknown and NTC are different from each other, and the primer dimer peak is not present in our unknown samples, can we accept the result?
hope anyone who's experienced in qRT-PCR can gv me some pointers here.. Thanks so much.
I think the first problem is the biggest one. whether or not you have gDNA after using that kit - mind you, no kit on the planet is always 100% - there may be DNA contamination from elsewhere, like perhaps in your buffer, water, or maybe amplicon contamination in your lab. this problem must be sorted out first
Thanks..So it means that the results can't be trusted? im not sure exactly what's causing the problem. maybe during cdna preparation or real time pcr set. but i've read in one review that if the ct for the no rt control is > 5 cycles than the cdna sample, than the result is acceptable. is it really/ or i should prepare everything all over again? the kit is the limiting factor. i dont have all that much left to finish the experiment..
it's entirely up to you, but I wouldn't trust it
I think that at least some of your data in the test samples is augmented by non-specific amplification. it's impossible to say how much, but again, I woudln't trust the data if it were me.
Thanx again aimikins..Looks like my experiment is really bad.. Now when i run my NTC control, i got the same peak too.. Maybe now the problem is not just in the sample, but maybe during the real time set up. I really dont know what to do. Now im just running my NTC control to see if there's s any weird peak that may pop up again! I have to finish my MSc as soon as possible..Any ideas on what i can do to solve this problem..
Before any RT reaction I have made it mandatory for myself to include a DNase treatment followed by a PCR which includes a positive control, only the RNA as template and no template control. This will tell if you have genomic DNA contamination in RNA or in the water or the buffer etc which you are using. Only upon convincing result I go for further real time experiments.