title: Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection.
abstract: The aim of this study was to evaluate a direct and automated post-polymerase chain reaction (PCR) detection system to simultaneously determine the relative gene expression levels of nine cancer-related human genes. Total RNA was prepared from flash-frozen biopsies derived from human colorectal tumors or normal mucosa and reverse-transcribed to cDNA which was PCR-amplified using primer pairs corresponding to the studied genes. In each reaction, the forward primer was labeled with a fluorescent dye. The PCR products were pooled and an internal size standard with a uniquely colored fluorescent dye was added. The samples were then subjected to automated capillary gel electrophoresis. Fragment analysis software was used to calculate the relative gene expression using beta-actin as the reference gene. We found that automated capillary gel electrophoresis with multicolor detection is a rapid, accurate and highly reproducible method for separation and quantification of PCR-amplified cDNA.
authors.affiliation: Department of Surgery, Sahlgrenska University Hospital/Ostra, Gothenburg, Sweden.
authors.forename: E
authors.initials: E
authors.lastname: Odin
authors.forename: Y
authors.initials: Y
authors.lastname: Wettergren
authors.forename: L
authors.initials: L
authors.lastname: Larsson
authors.forename: P A
authors.initials: PA
authors.lastname: Larsson
authors.forename: B
authors.initials: B
authors.lastname: Gustavsson
mesh_terms: D001322:Autoanalysis; D003106:Colon / Q000737:chemistry; D015179:Colorectal Neoplasms / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D007413:Intestinal Mucosa / Q000737:chemistry; D012007:Rectum / Q000737:chemistry; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013940:Thymidylate Synthase / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D013940:Thymidylate Synthase