Contamination issues - (May/27/2011 )
Hi to 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.
Following some discussions in this section, I found out that is doesn't matter if you get a +ve signal from the controls as long as they vary with the least concentrated unknown sample for at least 6-7 cycles? 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,
How ambiguous were the melting curves? Can you post a picture?
If the peak of negative controls is different (lower temperature) from the peak of samples, then you don't have contamination but probably template-dependent dimers. That usually means you can call them negative.
Yes, I am aware of that and the problem is that many times the peak in the -ve controls (no RT) was the same with the +ve controls. However, the ambiguousity lies in the fact that the "contamination" I supposedly have isn't consistent and let me explain what I mean.
I have used the same cDNA template in the -ve controls that I assesed for both my GOI and the reference gene. So, if I had a DNA contamination issue I would get a signal from both the wells with the GOI and the reference gene which I do not!
Furthermore, (and that is even more inexplicable) I went on and used the same cDNA template in a separate experiment I carried out just after the first, where I investigated a second GOI with the same reference gene (see design below). Have a look at my results:
no RT control (GOI1): clear no RT control (reference gene): after cycle 39/40
no RT control (GOI2): after cycle 35/40 no RT control (reference gene): after cycle 35/40
Do you think I can say that the noise from the no RT controls is non-significant if the Ct values between them and the unknown samples is more than 10?
Hope I did not confuse you with all that, the thing is that I am getting nuts with that!!!
So you have same cDNA and sometimes get amplification and sometimes not? The amplification can be dependent on the primers you use. Are your genes (GOI1,2, references) designed to be intron spanning?
Do you make replicates for each samples, are the replicates consistent? (like both two/all three are either negative or positive)
Yes exactly, that's the problem. My primers are designed to span introns and be in the CDS of the genes, but some of them form dimers or cross react between them (it was the price I had to pay for other parameters and I already knew that from their design). I always made duplicates but I treated them collectively in my analysis, so I do not know if they are all +ve or -ve. I can check it though.
However, how do you think that a primer can lead to amplification sometimes and sometimes not? What do you think about the Ct value difference safety range?
Intron spanning primers should not amplify DNA at all (depends on how intron spanning are they and how long is the intron) but sometimes they do, that could explain why some genes amplify -RT and some don't. This could be tested if you put DNA in your reaction and test on your primers. If there is an intron between primers however, the melting temperature should be different from the cDNA specific product.
However this doesn't explain amplification in NTC, that shouldn't amplify anything. If there is a product and has the same melting curve as cDNA product, then it's contaminated by cDNA. If the melting is different, it's dimers.
If you got stable contamination by specific product in all replicates but only in some genes and not the other, you may have contamination of reagents by a previous PCR product of that gene. Or contaminated pipette, if you ever use non-filtered tips or pipette the PCR product with the same one. If you have completely random positive calls (verified with specific melting) and not in all technical replicates, you may just cross-contaminated control wells with sample while pipetting. Especially plastic plates are prone to this, you should take extra care of your pipette movements if pipetting to the PCR plate.