The design of appropriate primers for the specific quantitative generation of DNA amplicons from diverse transcripts is an important requirement for most applications of real-time PCR. For the simultaneous measurement of multiple transcripts in a single experiment, an additional constraint, that all reactions proceed efficiently under the same conditions, must be imposed. In the first section of this chapter, we describe general primer and probe design guidelines for real-time PCR. In the second part, we present an online real-time PCR primer database encompassing most human and mouse genes identified to date. The primer database contains primers that perform well under a single set of conditions, allowing many simultaneous determinations of mRNA abundance to be carried out.
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