I performed a PCR on 25 samples in order to find a difference in the melting points by the mely curve analysis. I have divided my samples into three groups: homozygotes, heterezygotes and wildtypes. Now I have to calculate the intra and inter assay VC. Can someone help me out with this? Are there specific methodes to use to calculate the variations (SD/GEM*100)? What kind of pools do I need to set up to calculate the variations?
For my understanding, you would not be able to calculate intra and inter assay variation out of this data at all. Intra-assay variation means, that you repeat the measurement of the same (!!!) sample with the same test condition several times (n>2, better n>10) and calculate the variation between these values (still for the same sample!!!). Calculation of inter-assay variation is not always possible. You need a different assay system to compare with if possible, in your case a completely different method (which I can't think of) or at least a different software/PCR machine/different polymerase.
I guess what you want to know, is if homozygotic, heterozygotic and wildtype sample differ significantly in their melting point when using the same primers. I would look for the differences for each sample separately, for 25 samples it should be possible to see a tendency "manually" if there is any. If it's the same primer pair for all 25 samples, you might work with mean values and standard deviation for all samples within one group, but do not mix primer pairs for comparison.
It might help to find out how the intra-assay variation of your test system for each group is and compare that with the standard deviation within the different samples of the group. This tells you how big the random error of the assay is.
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