RAPID DETECTION OF THYMIDYLATE SYNTHASE GENE-EXPRESSION LEVELS BY SEMIQUANTITATIVE COMPETITIVE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION FOLLOWED BY QUANTITATIVE DIGITAL IMAGE-ANALYSIS

We describe a simplified and reliable polymerase chain reaction (PCR) method to quantify thymidylate synthase (TS) gene expression levels fr om clinical human tumor biopsy samples as small as 100 mg using the be ta-actin housekeeping gene as a reference standard. The semiquantitati ve RT-PCR is carried out by the coamplification of the target template and an external competitor using primer pairs common to both template s in the same reaction vessel. Quantitative digital image analysis is performed directly after electrophoresis, thus mRNA quantification is done quickly and without the use of radioactive substances. The observ ed relative TS gene expression levels varied between 3- and 40-fold, b ut most of the values were grouped within a 10-fold range. There is an observed 89% correlation between TS mRNA expression and protein level s. These findings suggest that preliminary experiments used to determi ne the linear range of RT-PCR amplification in noncompetitive semiquan titative PCR experiments, and the use of radioactive substances to qua ntify PCR products may be unnecessary.