Evaluation of a new high-dimensional miRNA profiling platform.
Evaluation of a new high-dimensional miRNA profiling platform.
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DOI：
10.1186/1755-8794-2-57
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发表时间：
2009-08-27
影响因子：
2.7
通讯作者：
Thibodeau SN
中科院分区：
文献类型：
--
作者：
Cunningham JM;Oberg AL;Borralho PM;Kren BT;French AJ;Wang L;Bot BM;Morlan BW;Silverstein KA;Staggs R;Zeng Y;Lamblin AF;Hilker CA;Fan JB;Steer CJ;Thibodeau SN
microRNA（miRNAs）是一类长约22个核苷酸、广泛表达的RNA分子，在真核生物中起着重要的调控作用。为了研究miRNA的功能，必须有定量其表达水平的方法。我们评估了一种新的miRNA分析平台，该平台利用Illumina现有的强大DASL化学作为分析的基础。使用来自5名结肠癌患者和4个细胞系的总RNA，我们评估了重复和不同量的输入RNA的miRNA表达水平的再现性。beta测试版本由Illumina的miRNA分析应用程序的735个miRNA靶标组成。同一平板内样品重复测定之间的重现性良好（斯皮尔曼相关性为0.91 - 0.98），不同日期运行的平板间重现性重复测定也是如此（斯皮尔曼相关性为0.84 - 0.98）。为了确定是否可以从宽范围的输入RNA获得质量数据，将从25 ng至800 ng范围内的量获得的数据与在200 ng下获得的数据进行比较。在RNA输入的范围内没有观察到影响。这些结果表明，非常少量的起始材料就足以使用Illumina miRNA高维平台进行敏感的miRNA分析。在重复测定之间观察到非线性偏差，表明需要进行丰度依赖性归一化。总体而言，Illumina miRNA分析系统的性能特征非常出色。
MicroRNAs (miRNAs) are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available. We evaluated a new miRNA profiling platform that utilizes Illumina's existing robust DASL chemistry as the basis for the assay. Using total RNA from five colon cancer patients and four cell lines, we evaluated the reproducibility of miRNA expression levels across replicates and with varying amounts of input RNA. The beta test version was comprised of 735 miRNA targets of Illumina's miRNA profiling application. Reproducibility between sample replicates within a plate was good (Spearman's correlation 0.91 to 0.98) as was the plate-to-plate reproducibility replicates run on different days (Spearman's correlation 0.84 to 0.98). To determine whether quality data could be obtained from a broad range of input RNA, data obtained from amounts ranging from 25 ng to 800 ng were compared to those obtained at 200 ng. No effect across the range of RNA input was observed. These results indicate that very small amounts of starting material are sufficient to allow sensitive miRNA profiling using the Illumina miRNA high-dimensional platform. Nonlinear biases were observed between replicates, indicating the need for abundance-dependent normalization. Overall, the performance characteristics of the Illumina miRNA profiling system were excellent.
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发表时间：
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影响因子：
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作者：
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通讯作者：
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