Determination of micro-RNA in cardiomyoblast cells using CE with LIF detection

Abstract

Micro-RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE-LIF) using fluorescence-labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA-499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA-499 was found when hybridization was conducted at 40 degrees C in 50 mM Tris-acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA-499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA-499 was completed within 1 h using CE-LIF. These results showed the potential of CE for fast, specific, and sensitive high-throughput analysis of low-abundance miRNAs in cell extracts, biofluids, and tissues.