Multiplex quantitative real-time PCR of laser microdissected tissue

This chapter describes a method for the rapid assessment of gene copy number in laser microdissected material using multiplex real-time polymerase chain reaction (PCR). Here a putative oncogene (ZNF217) was evaluated in a series of colon tumors, but the method is applicable to any locus for which a nucleic acid sequence is available. The preparation, laser microdissection, and optimum storage of snap-frozen tumor material from freshly resected tissue is described. A set of guidelines specific for real-time PCR assays is included to assist with optimum primer and probe design. In this assay multiplex real-time PCR was performed and our experience has demonstrated that a multiplex reaction allows for a more accurate assessment of gene copy number than a "singleplex" assay because it removes the need for an external control.