Rapid quantitative PCR determination of relative gene expression in tumor specimens using high-pressure liquid chromatography.
A reverse transcriptase polymerase chain reaction (rt-PCR) for quantification of gene expression has been optimized for analysis of folylpolyglutamate synthase (FPGS) and thymidylate synthase (TS), using beta-actin as an internal standard (house-keeping gene). Total RNA was isolated from tumor tissue, reversely transcribed to cDNA and PCR amplified with primers specific for TS, FPGS and beta-actin in separate vials. PCR products were separated and quantified by high-pressure liquid chromatography (HPLC) without addition of radioactive or fluorescent markers, which minimizes labor and occupational hazards. The day-to-day variation in the HPLC analysis was 2.7% and the within sample variations for rt-PCR/HPLC analysis of TS and FPGS were 18.5% for both assays. This method provides a tool for convenient gene expression analysis in clinical biopsies.
Odin
E
E
Department of Surgery, Göteborg University, Sahlgren's University Hospital, Sweden.
Larsson
L
L
Aram
M
M
Gustavsson
B
B
Larsson
P A
PA
D002851:Chromatography, High Pressure Liquid; D015179:Colorectal Neoplasms / Q000201:enzymology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D010453:Peptide Synthases / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012334:RNA, Neoplasm / Q000032:analysis; D015203:Reproducibility of Results; D013940:Thymidylate Synthase / Q000378:metabolism; D014158:Transcription, Genetic
D012334:RNA, Neoplasm; D013940:Thymidylate Synthase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase