ValidPrime correction causes missing data in reference gens

Here we discuss the problem of missing data

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ValidPrime correction causes missing data in reference gens

Postby Marusa » Wed Feb 03, 2016 2:57 pm


I have a problem when trying to do normalisation after correcting for gDNA. I have two reference genes, PPIA and RPLP, selected via Normfinder. I have two series of "identical" samples and when dealing with the first series, I had no problem. The second series had Cq values for PPIA quite high (29 or 30, this is 1 or two higher than my first series) and I guess this has something to do with the fact that when I correct with ValidPrime, some samples have a correction for more than 1, and some samples get a "NAN" in red. The rest of the genes are fine. If I then attempt to normalize to reference genes anyway, I get an error message "reference genes have missing data".
Can I somehow solve this without rerunning PPIA on the second set of samples? I have suspected a bit of a mastermix problem, but then I got nice looking curves, albeit late, so I thought it was ok after all. Maybe I was wrong and I'll redo it with a new mastermix, but in case I get the same problem later, I'd like to know what's going on.

Thank you!
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Re: ValidPrime correction causes missing data in reference g

Postby MultiDAdmin » Fri May 27, 2016 1:55 pm


Here is the cause rapported by user:

"we have found that the problem was in the very beginning – RNA quality. We assumed then the gDNA amount in the sample was actually higher than the intact RNA. "

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