I recently performed PCR and observed peaks for HEX and FAM, but the ROX signal was flat. I am unsure how to interpret and present these results.
Could someone please arrange a Zoom meeting to provide detailed guidance on this analysis?
Thank you for your time and assistance.
Dear Haris Ali Khan ,
I might be wrong, but I think there is a miss understanding with the usage of the dyes. I assume that you have used TaqMan assay for your gen of interest (GOI) which might be with probes in FAM and house keeping gene (HK) which might have bound to HEX. In some older PCR-cyclers (i.e. bio-rad icycler) specially those with a camera detection system you would need to make a flat field correction (meaning to correct for the lower excitated border wells compare with the stronger wells in the middle of the plate) that can be archived with a calibration plate or with another third dye. Therefore some suppliers are selling q-PCR mixes premixed or with individual packed ROX dyes to acquire a reference prior to the q-PCR.
So if you have not ordered an assay with probes bound to ROX everything might be right. If you have used a ROX bound probe to multiplex with two GOI and one HK I'm wrong and the expression of your gene is absent.
Best wishes Soenke
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