Moltzahn Felix, Hunkapiller Nathan, Mir Alain A, Imbar Tal, Blelloch Robert
The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, USA.
J Vis Exp. 2011 Aug 3(54):2552. doi: 10.3791/2552.
The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.
微小RNA（miRNA）广泛参与发育、组织稳态和疾病等关键过程，这引发了研究界和制药界的浓厚兴趣。为了研究miRNA，准确且可靠地定量微小RNA水平至关重要。通过比较野生型与小RNA缺陷型小鼠胚胎干细胞（mESC），我们发现先前发表的多重定量逆转录聚合酶链反应（qRT-PCR）技术缺乏准确性和可靠性。在此，我们描述了一种优化方法，包括在单重实时检测之前从先前的多重步骤中纯化去除过量引物，这显著提高了该技术的准确性和可靠性。此外，我们还解释了如何在纳升体积的微流控芯片上进行该技术，这可显著降低试剂成本，并实现高效的高通量miRNA表达谱分析。
