Dear all,
I performed several qPCR, which also worked fine, however, now during the evaluation I encountered an issue. My problem is that due to the restriction when choosing primers, I ended up with different lengthy amplicons, i.e. the endogenous control (S16) is 70nt, the target 1 is 131nt and the target 2 is 116nt long.
Regarding the fact that SyberGreen should "randomly" intercalate into my products, I assume that more SyberGreen intercalates in one product amplicon type 1 or 2 than in the endogenous control.Is it now a good idea to adjust the relative amount of expressed target (1 or 2) with a factor, e.g. (expression of target 1) * (70/131) and (expression of target 2) * (70/116)?
Further I have read that SyberGreen seems to prefer some sequences to intercalate, can/should this also be taken into account or simply ignored at all?
Kind regards.
If you run amplicon specific standards with your amplifications, you can calculate copy numbers directly from the regression curves and need not worry about sequence or size specific inconsistencies with your PCR.
You do not have to worry about amplicon length, because you are not looking at fluorescence levels per se but at the rise of fluorescence during every cycle. As long as your amplicon is not longer than 200 bp, it will double in amount during the exponential phase and this is the phase you use for quantification. As stated above, you correct for housekeeping genes to correct for variations in the amount of starting material between samples.
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