Name: high throughput microrna profiling: optimized multiplex qrt-pcr at nanoliter scale on the fluidigm dynamic arraytm ifcs
Uploaded: 2011-08-03T12:00:00
Duration: 7 min 27 s
Description: Watch this Scientific Journal Video about High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs at JoVE.com

我们想感谢Blelloch实验室评论文本。这项工作是由来自美国国立卫生研究院（K08 NS48118和R01 NS057221），加州再生医学研究所（CIRM）（种子补助RS1 - 00161，新教师奖RN2 - 00906）和皮尤慈善信托基金到RB和调频支持Wissenschaftlich Urologische GESELLSCHAFT EV。

<ol>
	<li>Mitchell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+as+stable+blood-based+markers+for+cancer+detection.">Circulating microRNAs as stable blood-based markers for cancer detection.</a> Proc Natl Acad Sci U S A. 105, 10513-10518 (2008).</li><li>Tang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=220-plex+microRNA+expression+profile+of+a+single+cell.">220-plex microRNA expression profile of a single cell.</a> Nat Protoc. 1, 1154-1159 (2006).</li><li>Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. 3, 644-645 (2003).</li><li>Suh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+function+is+globally+suppressed+in+mouse+oocytes+and+early+embryos.">MicroRNA function is globally suppressed in mouse oocytes and early embryos.</a> Curr Biol. 20, 271-277 (2010).</li><li>Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+ES+cells+express+endogenous+shRNAs,+siRNAs,+and+other+Microprocessor-independent,+Dicer-dependent+small+RNAs.">Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs.</a> Genes Dev. 22, 2773-2785 (2008).</li><li>Babiarz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNAs:+their+biogenesis,+regulation+and+function+in+embryonic+stem+cells.">Small RNAs: their biogenesis, regulation and function in embryonic stem cells.</a> StemBook. , (2009).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2008).</li><li>Wang, Y., Medvid, R., Melton, C., Jaenisch, R., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DGCR8+is+essential+for+microRNA+biogenesis+and+silencing+of+embryonic+stem+cell+self-renewal.">DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal.</a> Nat Genet. 39, 380-385 (2007).</li><li>Kanellopoulou, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicer-deficient+mouse+embryonic+stem+cells+are+defective+in+differentiation+and+centromeric+silencing.">Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.</a> Genes Dev. 19, 489-501 (2005).</li><li>Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom+microarray+platform+for+analysis+of+microRNA+gene+expression.">A custom microarray platform for analysis of microRNA gene expression.</a> Nat Methods. 1, 47-53 (2004).</li><li>Moltzahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+based+multiplex+qRT-PCR+identifies+diagnostic+and+prognostic+microRNA+signatures+in+sera+of+prostate+cancer+patients.">Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients.</a> Cancer Res. 71, 550-560 (2011).</li><li>Mestdagh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+universal+method+for+microRNA+RT-qPCR+data+normalization.">A novel and universal method for microRNA RT-qPCR data normalization.</a> Genome Biol. 10, R64-R64 (2009).</li></ol>

艾伦米尔是Fluidig​​m公司的雇员。否则，我们没有披露的财务利益。

miRNA是短（18-24个核苷酸），非编码RNA，从而调节基因表达的转录后由两个不稳定的信使RNA（mRNA）的抑制其翻译。6，至少有三分之一的人类基因组包含保守的miRNA约束力的网站在他们的3&#39;UTR和多能性，增殖和凋亡基因的miRNA明显的相互作用，表明在细胞命运的决定，组织动态平衡和癌症等疾病的重要贡献6,7因此，准确的microRNA表达谱广泛。兴趣。 为了测试发布的多重定量RT - PCR检测miRNA 表达谱协议2，我们作为阴性对照的小分子RNA的缺陷MESC。 DGCR8 - / -细胞缺乏规范的microRNA，而Dicer的- / -细胞缺乏规范和非canoncial micoRNAs 8,9 比较野生型水平的MES DGCR8 - / -细胞，我们发现几个表示没有检测到野生型细胞11 10小分子RNA的缺乏准确性。有的甚至呈相对较低的表达水平的基因敲除细胞（图3a）。我们推测，可能会造成大量的引物进行两个连续的多重步骤之前RT定量单一（RT和前PCR）缺乏准确性。然而，多重预扩增是必要的，以提高信号强度，尤其是当起始RNA浓度是有限的。净化远离本地聚丙烯酰胺凝胶电泳（图2）的大小选择的引物- PCR产物，我们可以证明，通过检测microRNA和假阳性信号的损失都DGCR8在精度显着改善 - / -和 Dicer - / -另外的背景（图3a和3b）修改后的QRT - PCR的方法，允许适当的分类DGCR8罕见的独立 / Dicer的依赖小分子RNA（ 图 3b ）11。因此，一个额外的步骤，以净化过量引物离预PCR产物是明显的优势，尤其是当使用大型复用底漆，每样本集。 预扩增周期的最佳人数是由输入RNA浓度。不能下或平衡overamplify应遵循的具体实验的详情。 与千余名已知的microRNA，使用标准的384孔板未必最广泛的microRNA分析，尤其是在比较多个样本。 Fluidig​​m公司的动态阵列国际金融公司使，用移液步骤和所需化学显着下降，以96对96纳升规模（6.7 NL）（9216反应）在一次实验中在不同的小分子RNA的个别样品测试。对于每一个运行的分析软件提供的扩增曲线，色码热图和循环阈值（CT）。同时大规模的分析，降低实验的差异，并允许平均表达式的值正常化，这出执行其他正常化战略，利用小分子RNA控制的。 相比市售384预先指定的TaqMan探针的良好平台，与定制引物的高通量分析平台的结合提供了实验的灵活性高。 优化的多重定量RT - PCR技术相结合的方式，用动态数组平台成功地允许我们同时屏幕改建48个前列腺癌患者血清中384的miRNA（图4）的水平，并没有控制穗（即合成的microRNA）的数据正常化。 11 尽管增加从样品制备到分析结果步骤，描述的方法是一个时间和成本效益的高通量方法简介miRNA表达水平的大样本集，即使在有限的起始原料。

high throughput microrna profiling: optimized multiplex qrt-pcr at nanoliter scale on the fluidigm dynamic arraytm ifcs

在底层开发，组织homoeostasis和疾病的关键进程的miRNA广泛参与，导致之间涌动的研究和制药界的兴趣。要研究的miRNA，它是必不可少的microRNA水平的量化准确，稳健的。通过比较野生型的小分子RNA的缺陷小鼠胚胎干细胞（MESC），我们揭示了在先前公布的多重定量RT - PCR技术的准确性和鲁棒性的缺乏。在这里，我们描述了一个优化的方法，包括净化远离以前的多重步骤之前单一的实时检测，大大提高了该技术的准确性和鲁棒性的过度引物。此外，我们将解释如何执行一个微流体芯片在纳升量的技术，大大降低试剂成本，并允许时间有效的高通量的miRNA表达谱。

Watch this Scientific Journal Video about High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs at JoVE.com

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

在这里，我们描述一个优化的多重逆转录定量PCR（QRT - PCR）结合起来作为一个时间和成本效益高通量的microRNA（miRNA）的表达水平的筛检工具的微流体平台的协议，尤其是当样本数量有限的工作。

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

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高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

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