A novel real-time PCR assay of microRNAs using S-Poly(T), a specific oligo(dT) reverse transcription primer with excellent sensitivity and specificity.
A novel real-time PCR assay of microRNAs using S-Poly(T), a specific oligo(dT) reverse transcription primer with excellent sensitivity and specificity.
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使用 S-Poly(T) 进行新型 microRNA 实时 PCR 检测，S-Poly(T) 是一种具有出色灵敏度和特异性的特异性 Oligo(dT) 逆转录引物
DOI：
10.1371/journal.pone.0048536
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Gou D
中科院分区：
文献类型：
--
作者：
Kang K;Zhang X;Liu H;Wang Z;Zhong J;Huang Z;Peng X;Zeng Y;Wang Y;Yang Y;Luo J;Gou D
MicroRNAs （miRNAs）是一种小的非编码rna，能够转录后调节基因表达。准确的表达谱对于理解mirna的生物学作用和探索它们作为疾病的生物标志物至关重要。设计了一种新颖、高灵敏度、可靠的miRNA定量方法，称为S-Poly(T) miRNA测定法。在该实验中，mirna通过S-Poly(T)引物进行聚腺苷化和逆转录，该引物包含一个通用的反向引物、一个通用的Taqman探针、一个寡核苷酸（dT）11序列和六个mirna特异性碱基。然后通过特定的正向引物和通用的反向引物扩增单个mirna，并用通用的Taqman探针检测PCR产物。S-Poly(T)分析显示，与茎环或基于poly(a)的方法相比，灵敏度至少增加了4倍。该方法在区分具有高序列相似性的mirna方面也具有显著的特异性。利用这种方法，我们分析了人肺动脉平滑肌细胞（HPASMC）中的mirna，并鉴定了9个与缺氧治疗相关的差异表达mirna。由于其出色的敏感性，正常人血清中循环mirna的数量从368个显著增加到518个。S-Poly(T)方法具有良好的灵敏度、特异性和高通量，为miRNA定量和鉴定组织或疾病特异性miRNA生物标志物提供了强有力的工具。
Background MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. Methodology/Principal Findings A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)11 sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518. Conclusions/Significance With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers.
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影响因子：
20.3
作者：
Diakos, Christofer;Zhong, Sheng;Wiemels, Joseph L.
通讯作者：
Wiemels, Joseph L.
影响因子：
11.2
作者：
Bader AG;Brown D;Winkler M
通讯作者：
Winkler M
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：
6.4
作者：
Brase, Jan C.;Johannes, Marc;Sueltmann, Holger
通讯作者：
Sueltmann, Holger
DOI：
10.1042/cs20100297
发表时间：
2011-03
期刊：
Clinical science (London, England : 1979)
影响因子：
--
作者：
Gui J;Tian Y;Wen X;Zhang W;Zhang P;Gao J;Run W;Tian L;Jia X;Gao Y
通讯作者：
Gao Y