I know usually the software does the calculation. However, I run into a problem with one of my tests. I had raw data, melt curve and PCR products (checked by gel). But, the amplification plot was blank, no curve. On the exported excel data sheet, the result, which was supposed to give me Ct value, was blank. I am trying to rescue the test result from the raw data, if there is a way.
It might be easier to find out what went wrong that your software does not recognize the amplification curve. Contacting the support for the instrument/software may be helpful.
Yue, this is correct in principle but omits some details about a possible reference dye normalization and baselin correction, detrending and so on.
Hi,
There is a software/tool you might abuse for your purposes, called linregpcr (1). It is designed for calculating intra-well amplification efficiency if you don't want to use a standard factor 2, but determine an empirical efficiency [see (2)]. Usually you would do a diltution series of an external standard (3), but if you have neglected to do so, you can also look at the amplification curves and fit a linear model.
But... you can skip all that and just use the Ct values it gives you from the raw fluorescence data if you want to save time. Although I must say that looking at all the individual curves via this software does make you get to know your primers.
Just google linregpcr for download link.
Note of caution: as Jochen said, in most standard mastermixes there is a correction for loading via a reference dye, you'll miss out on that. Hope your pipetting skills are up to scratch :-P
1. C. Ramakers, J. M. Ruijter, R. H. L. Deprez, A. F. M. Moorman, Neuroscience Letters 339, 62–66.
2. M. W. Pfaffl, Nucleic Acids Res. 29, e45 (2001).
3. R. Rasmussen, in Rapid Cycle Real-time PCR, Methods and Applications, Rapid Cycle Real-time PCR, Methods and Applications. S. Meuer, C. Wittwer, K. Nakagawara, Eds. (Springer Press, Heidelberg, 2001), pp. 21–34.
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