Hi there! Im running a qpcr right now. For negative control, I had two samples that is contain DNA template but supposedly undetected by my primer (I've checked this on primer BLAST) and NTC.
However, in the replicates I've got 2 wells out of 6 that is detected, one of them is ct 33 and the other is ct 36. If I decided to just rule this out as cross contamination, or might be primer dimer is that alright? I've read a journal that decided to make that ct > 30 in negative control means negative, but the thing is that from the standard curve I also want to obtain LOD result. And some of them got >35 in 0.0001 ng/uL result.
Does this means that I also have to rule everything including my positive sample which beyond ct > 30 means negative?
The thing is that the rest of the data is quite good and thats why if possible I wanna avoid having to re-do the qpcr.
Or... is that possible that primer could detect a sequence that is not listed on primer BLAST?
Hi Metty,
I wouldn't just set a random cutoff for a qPCR. The detection limit is depending on many factors, such as the intensity of your signal, or the age of your machine. If your signal develops at Ct 12, then a contamination in the NTC at Ct30 is negligible - if your signal comes at 28, then 30 is pretty close. Secondly, it is always possible that off target priming happens, even if primer BLAST says your primers should be fine. This is because the databases are never fully exhaustive and binding is not entirely predictable.
That being said, I would recommend to either do your qPCR with a melting curve (this way you can see how many products are forming), or, if you kept the plate and really cannot do the qPCR again, run the contents of the well on an agarose gel. This way you can distinguish the target amplicon and primer-dimers. If there are only dimers in your NTCs and no dimers in your other samples, the data should be usable.
Best
Carina
Carina Osterhof Thank you for your answer! I got ct of 16 in my positive sample. Unfortunately, i can't run a melting curve since I'm using a TaqMan probe. I do tried running them in a agarose gel, however I'm not sure if I accidentally made a contamination or something, but something wrong as all of my samples shows a band in DNA target, even the no detected ct one. Is there any way to prove this for taqman qpcr besides gel electrophoresis?
Sounds like you have contamination of that sample. Did you include a no-template control (just water)?
Those sort of uneven results in technical replicates can also be due to evaporation, issues with those wells in your machine, or pipetting skill.
What do you see in your standard curve replicates?
Katie A S Burnette thanks for answering! Yeah, I also include NTC, which turns out to be fine! My standards curve is fine. The thing is that I didnt checked them immediately after qPCR, I put them in 4C (not -20C) before gel which might be a mistake....?Cause I kinda notice an evaporation on the seal.. so I guess I cant rely on gel anymore to analyze the data...