Hi! I have to perform a qPCR experiment in which I would like to evaluate the expression of 13 genes (plus 3 HKG) on 10 samples. I must carry the runs on a 96-well qPCR plate since I need to compare the results with other runs performed before on that type of plate. So can I split the samples between plates if I use an interrun calibrator (i.e. a sample which is present in all of the plates), or is it better to keep the samples together even though that means only 2 genes will fit in each plate?
Hi Ot!
The best option is to choose "sample maximization".
So in your case indeed keep all your samples together but separate your genes over several plates.
In this case you don't have to use inter-run calibrators since all your samples that you want to measure/ compare for 1 gene are on 1 plate! You can compare the different genes with each other spread over different plates because little "differences" in Cq because measurements of the machine will not change the ratio between the values on the different plates
Laura
Ot Massafret I would recommend to distribute your sample based on genes. In each plate, you can do all samples of one test gene along with your best housekeeping gene. However, for this it is essential for you to first determine the best-fit reference/internal control gene. Then it would be straight forward to determine the relative quantification of each gene in all the samples.
Keep your samples together on one plate and array over multiple plates with inter plate calibrators
That is as you say 2 genes per plate with multiple samples
This is probably your most optimal configuration
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