Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection

The aim of this study was to evaluate a direct and automated post-polymeras e chain reaction (PCR) detection system to simultaneously determine the rel ative gene expression levels of nine cancer-related human genes. Total RNA was prepared from flash-frozen biopsies derived from human colorectal tumor s or normal mucosa and reverse-transcribed to cDNA which was PCR-amplified using primer pairs corresponding to the studied genes. In each reaction, th e forward primer was labeled with a fluorescent dye. The PCR products were pooled and an internal size standard with a uniquely colored fluorescent dy e was added. The samples were then subjected to automated capillary gel ele ctrophoresis. Fragment analysis software was used to calculate the relative gene expression using beta-actin as the reference gene. We found that auto mated capillary gel electrophoresis with multicolor detection is a rapid, a ccurate and highly reproducible method for separation and quantification of PCR-amplified cDNA. (C) 1999 Elsevier Science B.V. All rights reserved.