Hi, I would like to get any inputs on determining the lowest viral detection limits using real time PCR. My initial trials have failed to give an amplification plot in the software, but I see bands in the gel. Could it be because of a very small amplicon size (56bp)?
Thanks,
Maya
Hi,
56bp is very small- which detection strategy do you use? Probe-based or SybrGreen? Potentially the small product can be primer dimer not detected by the probe. Otherwise, if you use SybrGreen you can check if the detection temperature (72°C step) is to high for the small product (already melted?). Maybe you can run a 2-step PCR with the elongation (and detection) at the annealing temperature...
Good luck!
Thank you Muhammad and Norman for your valuable inputs. I will try these and see if I get any better results
Maya
Hi again, I tried as you suggested with a 2-step cycle and a standard curve with 10-fold dilutions. My results are strange in that the initial concentration failed to give amplification. Image attached.
Lower dilution gave a perfect 3.32 Ct difference. Image attached
I'd go with Normans answer - 56 BP is usually way too small to see on on a standard agarose gel electrophoresis, if you have a smudge below the 100BP marker that will almost certainly be primers/primer dimer - so your qPCR results probably indicate you have no product. We usually clone and sequence PCR products to confirm what they are before moving on to qPCR to be sure about this kind of thing. You might be better off designing alternative (amplify a larger amplicon) primers to resolve this as you are going to spend a lot of time faffing about otherwise. (are you sure you have RNA/viral RNA or DNA of whatever you are looking at in your sample in the first place?)
Thank you Rachael for your input. I am surprised with our results because the negative control is clean! Also, got the Ct difference in lower dilutions.
Maya
Hello Maya,
I feel a problem with primers. what is the size of your primers ?
Did you analyse them for self or heterodimers ?
http://eu.idtdna.com/calc/analyzer
What a researcher gave me as a test of non specific amplification is making a blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with an oligo composed by your frwd primer followed by the reverse complement of your reverse primer.
let's see
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