Regional Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic; Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 612 65 Brno, Czech Republic.
Regional Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic.
Anal Chim Acta. 2014 Feb 27;813:35-40. doi: 10.1016/j.aca.2014.01.023. Epub 2014 Jan 16.
Aberrant expression of microRNAs (miRNAs), short non-coding RNA molecules regulating gene expression, is often found in tumor cells, making the miRNAs suitable candidates as cancer biomarkers. Electrochemistry is an interesting alternative to current standard methods of miRNA detection by offering cheaper instrumentation and faster assays times. In this paper, we labeled miRNA in a quick, simple, two-step procedure with electroactive complex of osmium(VI) and 2,2'-bipyridine, Os(VI)bipy, which specifically binds to the ribose at the 3'-end of the miRNA, and hybridized such labeled miRNA with biotinylated capture probe attached to the streptavidin magnetic beads. Labeled miRNA was then detected at hanging mercury drop electrode at femtomole level due to an electrocatalytic nature of the peak from the Os(VI)bipy label. We obtained good selectivity of the assay using elevated hybridization temperatures for better discrimination of perfect duplex from single and double mismatches. After optimization of the protocol, we demonstrated feasibility of our assay by detecting target miRNA in real total RNA samples isolated from human cancer cells.
miRNAs（微小 RNA）是一类短的非编码 RNA 分子，能够调节基因表达，它们在肿瘤细胞中的表达常常出现异常，这使得 miRNAs 成为癌症生物标志物的候选者。与当前 miRNA 检测的标准方法相比，电化学方法具有仪器成本更低、检测时间更短的优势。在本文中，我们使用电化学活性的锇（VI）和 2,2'-联吡啶配合物 Os（VI）bipy 对 miRNA 进行了快速、简单的两步标记，该配合物能够特异性地与 miRNA 3' 端的核糖结合。标记后的 miRNA 与连接在链霉亲和素磁珠上的生物素化捕获探针杂交，然后在悬汞电极上进行检测，这是因为 Os（VI）bipy 标记物的峰具有电催化性质。我们通过升高杂交温度以更好地区分完美双链体与单链和双链错配体，从而获得了该测定方法的良好选择性。在优化方案后，我们通过检测从人癌细胞中分离出的真实总 RNA 样本中的靶 miRNA，证明了该测定方法的可行性。
