Analysis of data
The Ct value for an assay is related to the abundance of template in that reaction.
For the absolute quantitation method, a standard curve of a plot of Ct value against the log of the template concentration allows template concentration for unknown samples to be determined.
For the relative quantitation method, which is frequently used for mRNA analysis, the abundance of a transcript of a gene of interest is measured relative to that of an endogenously expressed gene such as actin or rRNA (housekeeping gene).
Relative quantitation works best when the genes being compared are amplified with similar PCR efficiencies, but corrections can be made if measured differences in efficiency are known.
We will give advice on appropriate experimental design and data analysis strategies, and how to set up spread sheets to process data.