So i'm testing samples for RNA viruses. We are using the phenol chloroform RNA extraction method, nanodropping to find concentration of RNA and quality, diluting the samples (in NF water) to 200 ng/microliter, converting the RNA to cDNA, then running q-PCR on each sample (in triplicate) with a no template negative control. All of the wells show DNA growth at various cycles. I had my mentor run everything from scratch as well, and he is also getting product in the NTC wells. Has anyone seen something like this happen before?
Hi,
First of, are you using a commercial kit for the PCR reagents or individual reagents that you add for the run?
It could be the case that one or two of your reagents used could be contaminated and you would have to rule out each one that you suspect. if you don't have the time and you have a fresh batch of unopened PCR reagents and of course sample volume to spare please try to run another round of PCR.
Does the curve of your NTC resemble that of your Positive control or any other sample? You would need to find out which of the reagents are contaminated. You can run the completed PCR products on an Agarose gel and check as well.
Hope this helps. Good luck
So I’m using Luna QPCR master mix, the weird thing is all of my positive controls aren’t looking good either, they are all over the place and nothing really looks like each other. I’m kinda leaning to primer dimer formation tbh, so I‘m thinking of doing a titration to see if a lower concentration of primer helps
Hi.
I have noticed a few points which might be of help.
First, what material are you using for isolating RNA? Is it possible some DNA is isolated as well?
If you are interested in cDNA, do you design primers that bind only to the coding sequence and not to introns? If not, then you should treat isolated RNA with DNase I to remove leftovers of DNA. Otherwise, you will have a signal in all wells.
Second is already mentioned contamination. Try to prepare small reactions with only one of the components being from a new, unopened vial. Then you can find out which component is contaminated. It happens very often with primer dilutions, so make a new dilution of primers. For this reason, it is very important to aliquot some reagents and use filter tips.
I guess you still have to optimize the PCR reaction if you say your positive controls do not look uniform. What is your positive sample anyway?
Lastly, do not use too much template nor primers. The less is better here.
Good luck!
The primers i'm using are from the literature, but we're looking at getting a new batch of primers in case that's the issue. For the amount of primers per well were only running 0.25 micromolars of forward and reverse primers and 40 nanograms of RNA per well. To be perfectly honest we really don't think its contamination, but were not using any DNase to remove genomic DNA. Because were looking for ss+RNA viruses I didn't thing DNA contamination would really affect the run. Today I'm about to do a complete negative control run with no template in the whole plate and try changing up different things to see if there is any evidence of contamination (pipettes, NF water, Stock primers, etc...) When i did a run the other day we got really great positive control samples so i dont think its pipetting error but just to be sure i'm running everything in triplicate.
Firstly, it is sure DNA interference with primers during PCR amplifications.
Also, you should add test negative control to the PCR run , it means add all components of the reaction except sample , use a dist water instead of sample, this is an evidence of there is no contaminations in the all reagents you used.
with my best wishes ....
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