Analysis of BART7 MicroRNA from Epstein-Barr Virus-infected Nasopharyngeal Carcinoma Cells by Capillary Electrophoresis
Analysis of BART7 MicroRNA from Epstein-Barr Virus-infected Nasopharyngeal Carcinoma Cells by Capillary Electrophoresis
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DOI：
10.1021/ac8014406
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发表时间：
2008-11-15
影响因子：
7.4
通讯作者：
Chen, Hua-Chien
中科院分区：
文献类型：
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作者：
Chang, Po-Ling;Chang, Yu-Sun;Chen, Hua-Chien
microRNA（miRNAs）是一类约22个核苷酸的非编码RNA分子，以序列特异性方式负调控其靶基因。在本研究中，荧光标记的反义DNA寡核苷酸与BART 7 miRNA在SSC缓冲液-十六烷基三甲基溴化铵（CTAB）中直接杂交，然后通过毛细管电泳与激光诱导荧光。CTAB介导的杂交允许探针在50.0 ℃下退火靶，这远低于计算机计算的66.4 ℃的解链温度。游离探针（22-nt）和探针/miRNA双链体（22-bp）可以在电渗流存在下用2%聚环氧乙烷与7 M尿素很好地分离。DNA探针迁移时间的重复性为10.66 +/- 0.34 min（n = 10），分辨率为1.12 +/- 0.11（n = 10），分离效率分别为1.71和1.74百万/米。探针/miRNA双链体的峰面积呈现良好的线性（r（2）= 0.9973）。此外，即使在存在2000倍过量的单核苷酸错配靶标的情况下，也未检测到假阳性结果。与其他方法相比，毛细管电泳不仅表现出优异的特异性，而且显示出可忽略的固有干扰，如人总RNA，初级miRNA或前体miRNA的影响。
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.