Absolute quant with TaqMan - Different RT & different plates? - (Jan/22/2015 )

Hello!
I am running absolute qPCR using Taq Man and running into possibly two issues:

1) Would running the standard curves and targets on separate plates be an issue? If yes, then how to avoid it and perhaps create a reference? Or maybe run e.g. three orders of magniture in the target plate, just to see if the efficiencies are the same?

2) My reference gene in plasmids has been made months ago and they were reverse transcribed using the regular RT (with oligo dT). I am using the high-capacity RT from different company (QScript from Quanta) that contains both oligo dTs and random primers. I think that would result in different efficiencies of RT reaction, so how to overcome this?

Thank you for help and sorry if this was previously answered. I tried to look it up :)

1) When running absolute quantification you need to have the standard curves on plate. For more samples, run a separate set of standards and analyse every plate separately.

Or, other possibility, if your cycler analysis module can do it, you may run a second plate with only one of the references, which will then be used as inter-run calibrator.

2) You need to keep the efficiency of standards and template the same if you want to have a corresponding copy-number. I remember for viral detection the standards were keeping deep frozen and used only for limited amount of time, as RNA degrades, and used in each amplification as an RNA template (same as the samples, which were RNA), so transcription was not only identical but also in the same time and conditions.

Depends why you need RNA standards, if only for efficiency correction and not an exact copy number (which is important for viral RNA quantification), you don't need the RNA standard at all, and just use cDNA in plasmid (which when linearized and mixed with a "dummy" rRNA to simulate similar complexity is enough for the efficiency correction).

The standards will of course create and arbitrary values, like "10 000 copies/reaction of linearized plasmid RNA" which doesn't correspond to real 10 000 copies/reaction of your sample RNA, but you can validate these values (i.e. 100 copies of standard is a threashold of clinical relevance of my RNA template) or just used them to compare inbetween samples.

RNA standards are tricky, and usually are used only when there is no better option, as RNA stability is low.