Analysis of BART7 MicroRNA from Epstein−Barr Virus-Infected Nasopharyngeal Carcinoma Cells by Capillary Electrophoresis

Abstract

MicroRNAs (miRNAs) are a class of approximately 22-nucleotide noncoding RNA molecules that negatively regulate their target genes in a sequence-specific manner. In the present study, a fluorescence-labeled antisense DNA oligonucleotide was directly hybridized with BART7 miRNA in SSC buffered-cetyltrimethylammonium bromide (CTAB), followed by capillary electrophoresis with laser-induced fluorescence. The CTAB-mediated hybridization allows the probe to anneal the target at 50.0 degrees C, which is well below the computer-calculated melting temperature of 66.4 degrees C. The free probe (22-nt) and probe/miRNA duplex (22-bp) can be separated well by 2% poly(ethylene) oxide in the presence of electroosmotic flow with 7 M urea. The repeatability of the migration time of the DNA probe was 10.66 +/- 0.34 min (n = 10), the resolution was 1.12 +/- 0.11 (n = 10), and the separation efficiencies achieved were 1.71 and 1.74 million per meter. The peak area of the probe/miRNA duplex exhibited an excellent linearity (r(2) = 0.9973). Furthermore, no false positive result was detected even in the presence of a 2000-fold excess of single nucleotide-mismatched target. Compared to other methods, capillary electrophoresis not only exhibits excellent specificity but also shows negligible effects of intrinsic interferences such as human total RNA, primary miRNA or precursor miRNA.