Hello everyone,
I have a problem with generating standard curves for qPCR with two distinct probes - first is FAM and second is HEX labelled. I'm trying to perform duplex PCR for 2 genes. So far I've performed singleplex PCR for both genes (using only 1 channel for detection) and the results were promising. The problem occurs during using two channels simultaneously (FAM and HEX). My thermocycler (LightCycler96, Roche) generates almost all the data - Ct, positive/negative call and a standard curve ...but ONLY for FAM labelled probes. Additionally, curves from HEX channel are visible as dotted lines. Do you have any ideas, what could be the problem?
I added screenshot from one of the experiments. It's a 2-fold standard curve, 2 genes simultaneously, 2 channels detection. Standard curve for FAM is visible, but for HEX the software could not generate it for some reason.
Hello Bin Mr ,
your suggestion was actually pretty usefull! Saving file and trying to generate another standard curve separately was not necessary, but I realized something else. I missed one checkbox in Sample Editor and correctly added concentrations for FAM, but HEX was left empty. Now everything works. Thanks for you reply!
For two calibration curves simultaneoulsy, at the Sample Editor tab all probes should be named at Gene, and standards should be marked as Standards at Sample Name window. Then concentration for each standard can be entered separately for each Gene, e.g. channel in Concentration window. If all information is entered correctly, two separate calibration curves will appear in Absolute quantification assay type.
Igor Oscorbin Thank you for your reply! It is precisely a solution for my problem, I just realized it a short time after writing a question here.
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