Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies

The archives of departments of pathology represent a unique source of morphologically defined biopsies derived from normal and pathologically altered tissues for which extensive clinical data are available. The exact quantification of nucleic acids in these biopsies offers a promising extension of current methodology to study the pathogenesis of many different diseases. The development of real-time PCR technology has greatly facilitated the realization of nucleic acid quantification. Now it is feasible to analyze large series of samples for the exact quantification of nucleic acids even if the number of target molecules is small and the amount of material available for analysis is limiting. This review focuses on our own experiences concerning the extraction of nucleic acids from fixed and embedded biopsies using both conventional approaches and laser-assisted microdissection and the subsequent application of real-time PCR methods for quantification of mRNA transcripts, gene copy number, and the methylation status. We provide a number of protocols to assist in the application of these techniques.