I am trying to get a duplex qPCR method to work. When running the two reactions (Gene, and ACTB-ref) in singleplex, the gene gives me a perfect sigmoid curve. The ACTB has a tendency to be "flat". However, when I run them in duplex the ACTB is even more flat. My question is if this is normal, and if I should expect a nice curve for both? If any of you have had a similar experience I would be glad if you could share your solution to this annoying "flat" ACTB (b-actin).
The flat appearance of the ACTB invariably means an incorrect primer or probe there - I would suggest re-checking your primer-probe sequences for your ACTB.
The ACTB, if truly an abundant reference gene in your system should be more of a reagent hog in general than your target -- unless your target is some flipped out amount of IL-2 or something like that. But, the fact that your ACTB looks unhealthy already in single-plex format smacks of a primer or probe sequence error... or too dilute primer-probe additions for ACTB if correct primer-probe sequences are being used for it.
I will get startet optimizing and looking at the assay again. Thank you for your suggestions and help Jack, it is very appreciated.
What do the raw Rn curves look like? The problem may be that you are trying to coamplify genes which have very different expression levels. If the software chooses a "baseline" range that is appropriate for the less expressed gene, it may cause the ACTB delta Rn curve to look strange. This may also be the case for ACTB alone if the baseline range is not adjusted properly.
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