Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada.
Anal Chem. 2011 Aug 15;83(16):6196-201. doi: 10.1021/ac2016213. Epub 2011 Jul 18.
MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naïve miRNAs.
微小 RNA（miRNAs）是小的（∼22 个核苷酸）调节 RNA，在癌症中经常失调，并已显示出作为组织和基于血液的癌症分类和预后的生物标志物的潜力。在这里，我们提出了一种使用单链 DNA 结合蛋白（SSB）和双链 RNA 结合蛋白（p19）作为分离增强剂的蛋白质促进亲和毛细管电泳（ProFACE）测定法，用于快速定量血清中的 miRNA 水平。该方法利用 SSB 与单链 DNA/RNA 探针的选择性结合或 p19 与 miRNA-RNA 探针双链体的结合。为了在没有聚合酶链反应（PCR）扩增的情况下检测血液样本中的超低含量 miRNA，我们应用 p19 涂覆的磁性珠离线预浓缩来自血清的合成 miRNA-122，然后在线在 ProFACE 测定中进行样品堆积。检测限为 0.5 fM 或 1 毫升血清中的 30,000 个 miRNA 分子，作为原始 miRNA 的潜在来源。
