This is an easy procedure of primer concentration optimization, where one is looking for the best possible conditions of amplification (lowest Ct). The procedure consists of varying the amounts of primer added to the reaction, in order to find the most efficient concentration, bearing in mind that 'more' does not always mean 'better'. There should be no difference in the other reagents used per reaction and the same input target cDNA should be used in all PCR reactions. For easy pipetting (for a 25 pl reaction), concentrations of 50, 100, 300, 500, and 1,000 nM can be used. An optimization matrix can be set up as shown below. Routinely, a simpler matrix (grey boxes) can be used to find good conditions and then further refined if necessary. An excess of primer, such as 1,000 F/1,000 R nM (as in the above examples) is often limiting and decreases the efficiency of the PCR reaction. An example of this procedure is illustrated in Figures 8.1A and 8.1B.
Following primer concentration optimization, a standard curve should be done, to perform an absolute quantification, but also to find the limits of sensitivity of the assay. A serial dilution of a factor 10 is easy to use and provides an obvious difference of Ct between points. A positive target sample should be used, which either contains a known amount of the target or is used as a default value of 1 (Figures 8.2 and 8.3).
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