Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.
Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.
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DOI：
10.1186/1471-2164-12-144
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发表时间：
2011-03-09
期刊：
影响因子：
4.4
通讯作者：
Jen J
中科院分区：
文献类型：
--
作者：
Jang JS;Simon VA;Feddersen RM;Rakhshan F;Schultz DA;Zschunke MA;Lingle WL;Kolbert CP;Jen J
microRNA（miRNAs）代表了一类不断增长的小的非编码RNA，它们是植物和动物中基因表达的重要调节因子。研究表明，miRNAs在人类肿瘤中发挥着重要作用，通过调控基因表达，影响细胞增殖和凋亡水平。目前，用于检测和测量miRNA表达的方法包括小通量和中等通量技术，例如标准定量PCR和基于微阵列的分析。然而，这些方法在用于大规模临床研究时具有几个限制，其中需要高通量和高定量技术来有效表征临床样品中的大量miRNA转录物。此外，存档福尔马林固定，石蜡包埋（FFPE）样品越来越成为基因表达研究的主要资源，因为新鲜冷冻（FF）样品往往难以获得，需要特殊的储存条件。在这项研究中，我们评估了FFPE和FF样品以及几种肺癌细胞系中的miRNA表达水平，采用基于高通量qPCR的微流控技术。将结果与使用相同样品的标准qPCR和基于杂交的微阵列平台进行比较。我们使用来自A549（R = 0.98; p < 0.0001）和H1299（R = 0.95; p <0.0001）肺癌细胞系的总RNA在miRNA表达的标准qPCR测定中证明了多重和单重RT反应之间高度相关的Ct值。由微流体技术（Fluidigm 48.48动态阵列系统）产生的Ct值与由ABI 7900 HT观察到的Ct值相比向更低的Ct值左移（平均差，3.79），表明微流体技术表现出更高的灵敏度。此外，我们表明，在FFPE和FF样品中，使用96多重RT反应，可以使用少至10 ng的总RNA来可靠地检测所有48或96种测试的miRNA。最后，我们比较了使用96.96动态阵列和Affytron微阵列通过qPCR测量FFPE和FF样品中的miRNA表达。两个平台之间可比基因的倍数变化比较表明总体相关性为R = 0.60。与96.96动态阵列检测到的13倍变化相比，由Affytron微阵列检测到的最大倍数变化为3.5倍。基于qPCR-阵列的微流体动态阵列平台可以与多重RT反应结合用于miRNA基因表达谱分析。我们表明，这种方法具有高度的可重复性，并且结果与现有的单重qPCR平台密切相关，其通量比常规测定高5至20倍，样品和试剂使用量约低50-100倍。我们建立了使用Fluidigm微流控技术的最佳条件，用于快速，成本效益高，可定制的miRNA表达谱分析和验证阵列。
MicroRNAs (miRNAs) represent a growing class of small non-coding RNAs that are important regulators of gene expression in both plants and animals. Studies have shown that miRNAs play a critical role in human cancer and they can influence the level of cell proliferation and apoptosis by modulating gene expression. Currently, methods for the detection and measurement of miRNA expression include small and moderate-throughput technologies, such as standard quantitative PCR and microarray based analysis. However, these methods have several limitations when used in large clinical studies where a high-throughput and highly quantitative technology needed for the efficient characterization of a large number of miRNA transcripts in clinical samples. Furthermore, archival formalin fixed, paraffin embedded (FFPE) samples are increasingly becoming the primary resource for gene expression studies because fresh frozen (FF) samples are often difficult to obtain and requires special storage conditions. In this study, we evaluated the miRNA expression levels in FFPE and FF samples as well as several lung cancer cell lines employing a high throughput qPCR-based microfluidic technology. The results were compared to standard qPCR and hybridization-based microarray platforms using the same samples. We demonstrated highly correlated Ct values between multiplex and singleplex RT reactions in standard qPCR assays for miRNA expression using total RNA from A549 (R = 0.98; p < 0.0001) and H1299 (R = 0.95; p < 0.0001) lung cancer cell lines. The Ct values generated by the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left-shift toward lower Ct values compared to those observed by ABI 7900 HT (mean difference, 3.79), suggesting that the microfluidic technology exhibited a greater sensitivity. In addition, we show that as little as 10 ng total RNA can be used to reliably detect all 48 or 96 tested miRNAs using a 96-multiplexing RT reaction in both FFPE and FF samples. Finally, we compared miRNA expression measurements in both FFPE and FF samples by qPCR using the 96.96 dynamic array and Affymetrix microarrays. Fold change comparisons for comparable genes between the two platforms indicated that the overall correlation was R = 0.60. The maximum fold change detected by the Affymetrix microarray was 3.5 compared to 13 by the 96.96 dynamic array. The qPCR-array based microfluidic dynamic array platform can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. We showed that this approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform at a throughput that is 5 to 20 times higher and a sample and reagent usage that was approximately 50-100 times lower than conventional assays. We established optimal conditions for using the Fluidigm microfluidic technology for rapid, cost effective, and customizable arrays for miRNA expression profiling and validation.
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