A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach.
A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach.
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通过优化的 RT-qPCR 方法量化 MicroRNA 的简便且特异的检测方法
DOI：
10.1371/journal.pone.0046890
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Han W
中科院分区：
文献类型：
--
作者：
Mei Q;Li X;Meng Y;Wu Z;Guo M;Zhao Y;Fu X;Han W
背景microRNAs （miRNAs）的时空表达模式对于验证其预测功能非常重要。RT-qPCR是公认的miRNA表达定量技术；然而，茎环RT-PCR和poly(T)-adapter assay这两种最常用的方法在实践中都不是很方便，特异性也很差。我们开发了一种优化的方法，集成了这两种方法，允许特异性和快速检测微量的样品RNA，并且相对于其他技术降低了成本。使用通用的poly(a)-茎环RT引物对同一样品的mirna进行多尿苷化并逆转录成cdna，然后用作SYBR®Green实时PCR的模板。该技术具有8个数量级的动态范围，灵敏度高达0.2 fM miRNA或低至10 pg总RNA。在这种高度特异性的分析中，在密切相关的miRNA家族成员之间以及与只有单个核苷酸差异的miRNA之间几乎没有观察到交叉反应。改良的RT引物的茎环结构的空间限制允许直接从细胞裂解物中检测mirna，而无需费力地分离总RNA，并且多(U)尾使得在同一运行中使用mRNA和mirna的多重RT反应成为可能。结论采用poly(A)-茎环RT引物对mirna进行RT- qpcr具有操作简单、特异性高的特点，这对于珍贵和/或难以获得的样品尤为重要。
Background The spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively. Results We have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run. Conclusions The cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.
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影响因子：
64.8
作者：
Lu, J;Getz, G;Golub, TR
通讯作者：
Golub, TR
影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
通讯作者：
Guegler KJ
影响因子：
50.3
作者：
Pichiorri F;Suh SS;Rocci A;De Luca L;Taccioli C;Santhanam R;Zhou W;Benson DM Jr;Hofmainster C;Alder H;Garofalo M;Di Leva G;Volinia S;Lin HJ;Perrotti D;Kuehl M;Aqeilan RI;Palumbo A;Croce CM
通讯作者：
Croce CM
影响因子：
2.7
作者：
Shi, R;Chiang, VL
通讯作者：
Chiang, VL
影响因子：
3.2
作者：
Veedu, Rakesh N.;Vester, Birte;Wengel, Jesper
通讯作者：
Wengel, Jesper