Based on the Rt-PCR analysis results of the oligos I have designed, it appears that there is a consistent signal from the NTC and negative patient samples with Ct values ranging from 30 to 32. However, I have noticed that in the positive patient samples, the signal sometimes resembles that of the NTC and sometimes it does not. I am curious about the possible reasons for these false positives in the NTC and false negatives in the positive samples. Could this be attributed to a faulty probe or potentially caused by dimerization?
is the NTC a no template control where you put in qPCR mix and primers and no cDNA, or is it a no transcription control where you did not add enzyme to your reverse transcriptase reaction and then add that as the template?
if it is the first one then you might have contamination in your primers or pipette. I would advise making the primers again fresh from stock and seeing what happens - run 4 wells to be sure.
clean your pipets with 1N NaOH or an RNase remover solution and rinse well with RNA grade water.
Julie Ann Dougherty thank you for your answer :) The NTC is negative template control for this case. However, in the case of contamination, what could be the reason for the absence of a signal in the positive wells? When I received a signal from the NTC, I changed the dH2O and 10 um oligos. Should not I need to get both positives to decide that contamination is the issue?
Your positives not amplifying is troubling too. Are you sure the patients are positive? or should they be positive? I would include a positive control for amplification in the set to ensure the plate was good. use either a sample that has worked before or PCR amplify some template to use. Also, it could be that your primers are just not good - it happens sometimes. they have all the right parameters but just don't amplify well. you could try designing primers to a different region of the gene. i usually design for the 5' end, middle and 3'end and test them to see which works best.
Julie Ann Dougherty The samples tested positive when I used a different commercial kit to check them. Since oligo sequences are acquired from the CDC's influenza assay, I assumed the sequence wouldn't be a problem. But I can't tackle the problem since I am unsure of it.
hmmmm.... I could be that your salt/buffer and primer concentrations differ too much from the commercial kit. do they include the full recipe after you buy it? or is it proprietary? You could have something inhibitor in one of your reagents. I would guess you are trying to save money and not use the commercial kit but if it has worked in the past then consider going back to it.
Julie Ann Dougherty No, unfortunately, I have no information about the contents of the other kit. Thank you very much for your answers :)
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