How do you lay out your PCR plates?

[disco volante]

When doing qRT-PCR, is it better to run each gene (housekeeping, and one or more experimental genes plus NTCs) on a 96 well plate and maybe only be able to fit six subjects on that plate or is it better to run one gene per plate but fit on all your subjects?

I've heard of people doing it both ways, but it seems more convenient if you run one gene per plate you can use the same housekeeping data over and over again without having to run it in parallel with every new transcript you decide to analyze. I am starting a PCR experiment and I want to make sure I lay my plates out properly.

[sunglassesatnight]

Definitely run one one gene per plate. If you run many genes but fewer samples per plate, you need to to interplate calibration on many plates, which can end up being a nightmare.

[JennYeap]

I did several plates for 1 gene due to the number of samples and replicates for that experiment including interplate control as calibrator without any nightmare. Main reason is the software i use for analysis of the expression- qBase plus software programme. The software auto-calibrates the variation in different plates. alternatively, you can manually adjust the Ct values of your interplate controls so that they have similar Ct values for each plate.

[sunglassesatnight]

Yes JennYeap you are right. I have also used multiple plates because of large sample size, but without qbase. It's not difficult when you have to calibrate between two or even three plates, but if you have 30 samples on different plates, it quickly turns into a gongshow of interplate calibration madness.