Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6.
Anal Biochem. 2011 May 15;412(2):165-72. doi: 10.1016/j.ab.2011.01.030. Epub 2011 Feb 1.
MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using p19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating p19's utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1fmol and a linear dynamic range from 1pmol to 1fmol.
微小 RNA（miRNAs）是存在于所有后生动物中的内源性转录后调控因子，在几乎所有细胞过程中都发挥着关键作用。它们的异常表达与几种疾病状态有关；因此，能够灵敏和特异性地分析 miRNA 微环境的技术将在预后、诊断和治疗中有重要应用。本文介绍了一种使用 p19 快速定量 miRNA 水平的方法，p19 是一种源自弹状病毒的 RNA 干扰抑制剂，对 19 个碱基对的 RNA 双链具有序列非依赖性和大小选择性亲和力。我们提出了一种基于表面等离子体共振（SPR）的 miRNA 传感方法，其中 RNA 探针固定在金表面上，证明了 p19 在识别 miRNA 结合探针方面的实用性。这使得能够在纳摩尔级的低浓度下检测 miRNA。为了提高灵敏度，还进行了基于珠的酶免疫测定，该技术的检测下限为 1fmol，线性动态范围为 1pmol 至 1fmol。
