Hi everyone,
I am applying RT-PCR using SYBR GREEN and i am really worried whether I have contamination in my samples or not. This is why I also run no RT controls and a NTC for both my GOI and the reference gene. My runs last for 40 cycles and I usually get a positive signal after cycle 35, either from the no RT controls or the NTC.
I found out that is doesn't matter if you get a +ve signal from the controls as long as they differ for at least 6-7 cycles with the least concentrated unknown sample. Is this a general rule I should look for or are there any other criteria I should know? I used to check the relative quantity of the samples and compare it to that of the controls or looked at the melting curves to see if the controls exhibited the same melting pattern with the unknown samples. The findings were ambiguous so I would appreciate any help,
Theodore
Thanks very much Wilco! This is where I finally got to. However, how would you define ''big enough''? I think Ct difference>5 is sufficient but I doublecheck with the relative quantities and make sure the unknown are 100-1000 times bigger than the no RT or NTC controls? Do you think that's enough?
Dear Nor Asfahani Ideris,
no RT stands for no Reverse Transcriptase controls. I have extracted RNA from my tissue, do Reverse Transcription and then use the cDNA for RT-PCR. Indeed, and thanks to a post I read on this site, it is possible that my RNA extraction with Trizol reagent is not that effective and my samples a re contaminated with DNA from the beginning (but I cannot do anything for that now).
The problem with the +ve controls is that, for instance, they have a Ct value, say cycle 36 and an unknown sample has a Ct values of 32? How could I trust such results? And what really annoys me is that sometimes they give the same melting profile with my unknown samples (suggesting that there is DNA contamination). This is why I want to use the relative difference in quantities; so that I will not care whether there is actually contamination or not.
I have run te gels and have optimised the annealing temperature for my RT-PCR (54 C). The bands were unique and clear so I trust there is not an issue with my thermal profile but rather a contamination at my RNA or environmental contamination during loading the plate (I tried to cover everything when I load the plate with slightly better results).
It is now too late to change anything as I finished my experiments and I need to interpret the data. So, my concerns lay on the fact that I am afraid sometimes to count one sample value and need to know when I can do so (and for future experiments I am not going to do the same mistake again, I will always use the safer probes).
Thanks
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