Design of primers and probes has been described elsewhere (see Chapters 5 and 7). The value of standardized reagents cannot be stressed highly enough (Gabert et al., 2003). However, where this is not possible, sequencing and design of patient specific reagents can be successful. Sufficient template should be added to ensure adequate sensitivity. The number of replicates required varies with application; however most laboratories would consider three to be the minimum for accurate quantitation and removal of outliers. The definition of replicate also varies; some centers process separate aliquots of cells, but it is more common to process one batch and set up multiple wells adding DNA or cDNA to the individual wells. If only one replicate is positive with a high Ct, the result should be confirmed using more replicates.
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