REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION-BASED METHODOLOGY TO QUANTIFY DIFFERENTIAL GENE-EXPRESSION DIRECTLY FROM MICRODISSECTED REGIONS OF FROZEN TISSUE-SECTIONS

Quantitative differences in the expression of oncogenes are a critical feature of the cancer process. Several methods are currently availabl e for assessing differential gene expression, but none can be used to determine quantitative changes in gene expression from small numbers o f cells. The ability to conduct this type of quantitative analysis wou ld be useful in the study of definable, early stages of carcinogenesis when very few cells are involved. We therefore developed a highly sen sitive, slide-based technique that incorporates the benefits of in sit u polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PC R) to quantify differential c-myc gene expression from liver tissue se ctions having either low or high levels of proliferating hepatocytes. To eliminate the need for isolating and quantifying mRNA, cells of int erest were microdissected from frozen histological sections and their RNA directly subjected to RT-PCR amplification. These reactions were c onducted in the presence of an internal RNA standard that was specific ally designed to normalize differential RT and PCR efficiencies betwee n samples. GENESCAN software analysis was used to determine the ratios of the RT-PCR products of the target gene to the RNA standard. These ratios were then normalized to the numbers of cells isolated, as quant ified by image analysis, and comparative gene expression values were d etermined between sample groups. We conclude that this technology can be adapted to study any gene of interest in any type of frozen tissue or isolated cells. This methodology is particularly applicable to the molecular analysis of histopathologically distinct preneoplastic and n eoplastic lesions identified on tissue sections. (C) 1997 Wiley-Liss, Inc.