Im doing a qpcr for genotyping ERCC1 rs11615, now Im in a middle of optimization of Probe ERCC1 wild type, with 2 confirmed sample (WT and heterozygous sample).
and the result, Ive got a big CT value (more than 38) in WT sample, and no ct in every heterozygous sample. what does it mean? and is that valid result or I have an error in doing a lab skill?
Thankyou
Dear Nadiya,
Cq of 38 has to be ignored so in total you do not have amplification in the both samples.
I agree with Mohamad. You should get ct value lower than 30, better lower than 25 to get interpretable results.
Check the PCR products on a gel.
Do you have a positive control reaction (e.g. a house-keeping gene)? If not, use one (at least). Using SYBR Green, do you get amplification curves with lower ct values? Does it still word when you add the probes to the SYBR Green assays?
If this is genotyping, it should be fairly straightforward.
Do you know your primers work?
If "no", then optimise your primers using clean gDNA of known genotypes and establish what sort of Cq values are associated with positives and negatives (for probes, negatives should be essentially "no amplification").
If "yes", then what sort of values do you typically see? Could it be that you are using
- not enough gDNA
- too much gDNA
- incredibly dirty gDNA
?
All of these things can be inhibitory/detrimental to PCR. Not enough target means you deal with stochastic effects (sometimes there's no target, sometimes there's some: high variability, high noise). Too much means you inhibit the reaction by saturation, and might also include excess contaminants that impair reaction efficiency. And dirty DNA is just...well, generally bad. Usually DNA is more forgiving than RNA>>cDNA, because genomic DNA extraction doesn't use quite as many horrible chemicals and solvents, but it can still be bad, and it might be worth checking just in case (nanodrop or equivalent).
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