Semi-quantitative detection of RAGE cDNA by electrochemical biosensor using HOECHST 33258

Abstract

Electrochemical biosensor has been applied for detection of gene expression of β-actin and RAGE genes. Using linear sweep voltammetry (LSV), β-actin expression in HeLa cell line and RAGE gene expression in HepG2 cell line were detected from the anodic current peak and the results were compared with the conventional agarose gel electrophoresis method. In brief, mRNA was reversed to cDNA and amplified by PCR, the PCR products was subjected to detection either by the electrophoresis or electrochemical biosensor methods. Precision of the biosensor technique was acceptable (β-actin: CV= 1.88% for 10⁴ copies and 4.68 % for 10⁹ copies; RAGE: CV = 2.25% for 10⁹ copies, and 3.74% for 10 copies). In biosensor technique, the PCR products were measured in the same run with various concentrations of standard, and copy number of β-actin gene was interpolated from standard curve. Copy number of β-actin gene was then compared between the two techniques. At 95% confidence limit, the two methods had no significant difference and had significant correlation (y = - 40383.0623 + 1.0233 x; p>0.10). Biosensor method was more sensitive than the conventional electrophoresis method because it could detect RAGE gene as low as 10 copies while the conventional method could detect visually at over 10⁴ copies and the linearity for semi-quantitative measurement started from 10⁶-10⁹ copies. When the electrochemical biosensor was applied to detect RAGE gene expression, we found that RAGE gene was expressed twice more in HeLa than HepG2 (relative value of 0.000905 vs. 0.0004670).