Dear all,
I am planning a qPCR experiment with multiple samples (4 groups with 7 samples + a dependent control to each sample and an undipendent control group, 7 samples again ) and multiple target genes (about 20). Usually I try to mix each plate with samples of different groups, which is obviously not possible with this experiment. Does anyone have suggestions how to do the plate design?
Thanks
If you can neither run "all genes" (per sample) nor "all samples" (per gene) on a single plate, then you will have to yous some inter-plate calibration (run a series of the sample standard/sample [so-called IPCs] on each plate).
https://de.slideshare.net/idtdna/high-throughput-qpcr-tips-for-analysis-across-multiple-plates
If you can neither run "all genes" (per sample) nor "all samples" (per gene) on a single plate, then you will have to yous some inter-plate calibration (run a series of the sample standard/sample [so-called IPCs] on each plate).
https://de.slideshare.net/idtdna/high-throughput-qpcr-tips-for-analysis-across-multiple-plates
Hi,
Other option is having some common samples (measuring the same gene) between each couple of plates and, after running the plates, adjust the threshold of the second plate to have the same (or very similar) ct values for the common samples in both plates.
Another option is to use 384-well plates although you need a different machine to run them.
good luck!
Hi, thanks for your answers!
I feel the inter- plate calibrator would be a great option, but unfortunately it wasn't possible in this experiment ( I think I will use one in the future), just as I don't have the possibility to use a 384 well plate. I started the experiment now using 1 sample of each group per plate with the housekeeping and 3 target genes tested per plate. This means to test all samples it needs 7 Plates (and each Plate has one sample of each condition on it, so they are distributed evenly on the different plates). After that I start all over again with 3 new target genes and of course the housekeeping gene again. If I understand Jochen Wilhelm and the link right this would be the "all genes per sample solution" and should be all right? I am using the same Threshold for all genes and across all plates (very similar for the automatic set up anyway). I am just wondering if it is necessary to set the baseline manually equal for the same genes across the plates, too. It would be great if somebody could give me advice on that matter.
Thanks again,
Lena
Sounds ok.
PS: It's not required (but ok) to have the same threshold for all genes, not even for the same gene across the plates. However, the difference in the thresholds between target gene and reference gene must be identical across plates (otherwise, dCt values from different plates would not be comparable).
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