Liquid Chromatography Based Multiplex Gene Expression Analysis Using Fluorous Separation Technique with Fluorescence Detection

Abstract

We have developed a comprehensive method for analyzing gene expression levels by combining the fluorous-fluorescence hybrid probes, oligonucleic acids modified with a perfluoroalkyl tag and fluorescent probes, with the fluorous liquid chromatography (LC) – fluorescence detection. When Taq polymerase chain reaction (PCR) was performed using this probe, fluorescent mononucleotides were released according to the amount of template gene targeted. Because each probe differs in the linker length of the fluorescent probe and the type of nucleobase, multiple gene expression levels can be analyzed by LC as the amount of fluorescent mononucleotides produced. The excess unreacted probe, which did not undergo the Taq PCR reaction, was selectively removed by adsorption in the fluorous LC column introduced as a pretreatment column. The method established in this study allows simultaneous LC quantification of three housekeeping genes. In addition, LC separation conditions for the simultaneous analysis of nine gene expression levels were established, and comprehensive detection of cytokine genes in lipopolysaccharide-stimulated leukocytes was successfully achieved.