Target and reference genes

[tiago]

Hi Mikael!
My study is about relative gene expression. I have 7 samples and 10 genes target. I used to do three biological replies and three technical replies. Actually I am looking for the best reference genes for this study (using 8 reference genes candidates)….after, when I get the betters reference genes I going to do the relative gene expression, so:
1) Should I have to run my reference genes (2 or 3) in all plates together with my genes target? If yes, how can I organize then in my plate (96 wells)? There is no space for all…If I needed run my reference genes in all plates, how can I do to put the “ct” of reference genes in the GenEx (Excel table), because I will have a lot of “ct” from reference genes?
2) And about interplate calibrator (In this case)?

Thanks.

[Mikael Kubista]

In general when both technical and biological replicates are available, the technical replicates shall be averaged and statistics (in this case Normfinder) shall be applied on the biological replicates.
You need to measure reference genes in all samples, of course. If you can plate such that all genes in every sample is measured on the same plate you don’t need interplate calibration. If this is not possible, you should have interplate calibrator. An interplate calibrator is a robust sample run preferably in at least triplicate on each plate and analyzed for an arbitrary target using the same instrument channel as for the analyzed genes. Soon a viewlet on Interplate calibration will be available on:

http://www.multid.se/tutorials.php

Good luck!