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Determination of micro‐ RNA in cardiomyoblast cells using CE with LIF detection
Author(s) -
Ban Eunmi,
Chae DongKyu,
Song Eun Joo
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200442
Subject(s) - rna , microbiology and biotechnology , chemistry , messenger rna , cell , biomarker , rna extraction , oligonucleotide , gene expression , dna , biology , biochemistry , gene
Micro‐ RNA s (mi RNAs ) are small, endogenous, singlestranded, and noncoding RNA s. The mi RNA s have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating mi RNA s 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 mi RNA in cell extracts. The target mi RNA is mi RNA ‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of mi RNA , we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized mi RNA ‐499 was found when hybridization was conducted at 40°C in 50 mM T ris‐acetate (pH 8.0) buffer containing 50 mM N a C l, and 10 mM EDTA for 15 min. The hybridized mi RNA ‐499 was detected in cultured H 9c2 cardiomyoblast cells and the analysis of mi RNA ‐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 mi RNA s in cell extracts, biofluids, and tissues.