Absolute quantification determines the exact number of the target present in the sample, by relating the PCR signal to a standard curve, therefore requiring that the absolute quantities of the standard be known. Absolute quantification makes it easier to compare data from different assays and necessary when exact transcript copy number need to be determined, e.g. viral or bacterial load in microbiology clinics (Mackay, 2004; Livak and Schmittgen, 2001).
The relative quantification method quantifies the target sequence amount normalized to an endogenous reference control (Livak and Schmittgen, 2001). This reference can be co-amplified separately or in the same tube in a multiplex assay; therefore with this method, rather than using a known amount of standard, comparison of the relative amount of target sequence to the reference values is possible (Livak and Schmittgen, 2001). The result is given as relative to that reference value and reference can be any transcript, as far as its sequence is known. Housekeeping genes are often used for this purpose, as they are widely expressed in various cells and tissues. The comparative Ct method is similar to the relative quantification method, except the usage of arithmetic formulas to achieve results for relative quantification. The relative standard or comparative Ct methods are simpler and easier to establish than absolute quantification as well as providing sufficient information in many cases. Details of quantification methods are given in Chapters 3 and 6.
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