Manuel Bueno wrote:
> We are using qBASE and geNORM for our qPCR
> experiments.
>
> We are just starting to have some bigger projects,
> i.e. projects, where we have to use several 384 well
> plates within one experiment. Concerning this I have a
> few questions.
>
> For one experiment we can fit all samples on one
> plate, but have to split the genes (as yo have
> recommended in the workshop in Freising). We have 3
> reference (houskeeping) genes on plate one and some
> target genes. The other target genes have to be run on
> plate 2 and 3, but of course normalized with the 3
> reference gens on plate 1. In this case, where the
> samples are always the same on all plates, do we have
> to use an interplate calibrator?
No.
> Or is it valid to
> calculate the deltaCts for the target genes on plate 2
> with the reference genes on plate one without doing an
> interplate calibration?
Indeed. (qbase should take of the calculations though...)
> Some colleagues even think it
> would be necessary to repeat the 3 reference genes on
> each plate...is this necessary?
>
No.
>
> For another experiment we have to split the samples
> between plates, because there are too many. In this
> case we certainly have to use a calibrator sample. Or
> better 3?
>
Yes. We recommend at least 3 inter-run calibrators. Apart from unknown
samples, you could also use the standards for inter-run calibration
(provided they are the same on both plates).

Good luck!

Jo vandesompele
> Thanks a lot in advance for your help
>
> kind regards
>
> Johann and Manuel