Multiplex Quantitative Real-Time PCR of Laser Microdissected Tissue

  • H. Patrick
Part of the Methods in Molecular Biology™ book series (MIMB, volume 293)

Abstract

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.

Key Words

Colon cancer quantitative real-time PCR ZNF217 gene copy number laser microdissection multiplex PCR ABI7700 sequence detector 

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Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • H. Patrick
    • 1
  1. 1.Department of PathologyUniversity of AberdeenAberdeenUK

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