Handling PCR data requires some care. Typically the user first gets Ct values that are 'normalized', e.g. with the gNorm method (essentially this implies selecting one or several housekeeping genes and computing normalization factors based on these). The thus normalized Ct values are called delta-Ct values (i.e. they are the difference between Ct for each gene and Ct for the housekeeping genes). It is also possible to compute delta-delta-Ct values by comparing the delta-Ct values for a sample with those for a calibration sample.
Also note that the higher the Ct value, the lower the gene expression level. Thus, what the instrument manufacturer denotes 'fold change' might be the ratio between genes and houskeeping genes, i.e. delta-Ct values.
More details are available in this (How to analyze qPCR data B.pdf) document. |
