Rapid Detection of Thymidylate Synthase Gene Expression Levels by Semi-Quantitative Competitive Reverse Transcriptase Polymerase Chain Reaction followed by Quantitative Digital Image Analysis

Abstract

We describe a simplified and reliable polymerase chain reaction (PCR) method to quantify thymidylate synthase (TS) gene expression levels from clinical human tumor biopsy samples as small as 100 mg using the Β-actin housekeeping gene as a reference standard. The semiquantitative RT-PCR is carried out by the coamplification of the target template and an external competitor using primer pairs common to both templates 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 observed relative TS gene expression levels varied between 3- and 40-fold, but most of the values were grouped within a 10-fold range. There is an observed 89% correlation between TS mRNA expression and protein levels. These findings suggest that preliminary experiments used to determine the linear range of RT-PCR amplification in non-competitive semiquantitative PCR experiments, and the use of radioactive substances to quantify PCR products may be unnecessary.

Rapid Quantitative PCR Determination of Relative Gene Expression in Tumor Specimens Using High-Pressure Liquid Chromatography

“…By using several dilutions of the reference gene, the absolute concentration of target cDNA is established when the amplification of both genes are equal. However, it is extremely difficult to find synthetic genes that are amplified with the same efficiency as the target gene, and the efficiencies decrease in a nonequivalent manner at different starting concentrations of the genes [6,7]. This method also requires gel electrophoretic resolution and densitometric analysis of two closely migrating DNA fragments after completion of the PCR, which implies considerable manual dexterity and makes it less suitable for routine use.…”

Detection of Thymidylate Synthase Gene Expression Levels in Formalin-Fixed Paraffin Embedded Tissue by Semiquantitative, Nonradioactive Reverse Transcriptase Polymerase Chain Reaction

“…The use of digital image analysis (DIA) following silver nitrate staining of polyacrylamide gels to di rectly quantify the PCR products eliminates the need for the use of radioactive substances in quantitative RT-PCR. Quantitative DIA (Q-DIA) enables the rapid and direct quanti fication of the concentrations of amplifica tion products [15,21,22], Furthermore, Q-DIA has recently been used to compare the observed level of TS gene expression relative to intracellular enzyme activity levels in shock-frozen clinical biopsy specimens of normal and tumor tissues [23], In the present study, this new method of Q-DIA analysis will be coupled with a novel method of mRNA extraction from formalin-fixed paraf fin-embedded samples, to analyze TS gene expression levels for normal and tumor biop sies from the same patients with the hope of gaining retrospectively insight into mecha nisms of tumor progression and resistance to chemotherapy.…”

Sonification as a Means of Enhancing the Detection of Gene Expression Levels from Formalin-Fixed, Paraffin-Embedded Biopsies

“…The use of RT-PCR provides a means by which the sensitivity of PCR in the detection of specific genes can provide many advantages over previous methods, especially in gene expression studies (2,4,(7)(8)(9)(10)(11). By modifying the method of mRNA extraction outlined by de Andrés et al (3) by the addition of sonification, it has been shown that archival biopsy sections can be used as a source of intact mRNA for retrospective gene expression studies.…”