can you go into more detail how you have spread your samples on the two plates? from one sample, you have the reference gene and all your target genes on one plate but you have so many samples that they are spreaded on two different plates? Moreover, you have the same calibrator sample one every plate?
Concerning your question about the Ct range: this is not simply to answer and I disagree that the Cts have to be around 20. This is very low and I achieve this only for very high expressed genes like ACTB. If you want to do it correctly, you have to do a standard curve to determine the linear dynamic range of your assay. Then you can analyze all your samples which Cts are located within that linear range.
This is just a short explanation of a detail within a complex method, but if you are interested and want to know more you can download the literature in the pinned thread. I think many of your questions will be answered there and if something is unclear do not hesitate and ask here again.
just one more comment: if possible, always try to avoid inter-run calibration. always try to pack as much samples as possible on one plate. let's say for a 96well plate with duplicates it would be 47 samples and one water control for one target gene (eg. gene "A"). you would do a second plate with your reference gene (eg. 18SrRNA). then you don't have to do a calibration between the plates because you only compare your samples for gene A expression which are all together on one plate. although you normalize with your reference samples which are on a different plate the technical variation between the two runs (one plate gene A and one plate 18S) doesn't concern you because the relative 18S expression differences within your samples are not affected by this.
this approach also saves expensive reagents.
If you have more than 47 samples which you have to split between plates then try to use more than one inter-run calibrator (IRC) because one failed IRC on a plate can ruin all the other data.
Edited by tea-test, 07 March 2009 - 11:45 AM.