High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
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DOI：
10.3791/2552
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发表时间：
2011-08-01
影响因子：
1.2
通讯作者：
Blelloch, Robert
中科院分区：
文献类型：
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作者：
Moltzahn, Felix;Hunkapiller, Nathan;Blelloch, Robert
miRNAs在发育、组织稳态和疾病的关键过程中的广泛参与引起了研究和制药界的极大兴趣。为了研究miRNA，microRNA水平的定量是准确和稳健的。通过比较野生型与小RNA缺陷型小鼠胚胎干细胞（mESC），我们揭示了先前公开的多重qRT-PCR技术缺乏准确性和稳健性。在这里，我们描述了一种优化的方法，包括在单重真实的时间检测之前从先前的多重步骤中纯化掉过量的引物，这显著提高了该技术的准确性和鲁棒性。此外，我们解释了如何在纳升体积的微流控芯片上执行该技术显着降低试剂成本，并允许时间有效的高通量miRNA表达谱。
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.