Real-time quantification of microRNAs by stem-loop RT-PCR.
Real-time quantification of microRNAs by stem-loop RT-PCR.
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DOI：
10.1093/nar/gni178
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发表时间：
2005-11-27
影响因子：
14.9
通讯作者：
Guegler KJ
中科院分区：
文献类型：
--
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
一种新的microRNA（miRNA）定量方法已经开发出使用茎环RT随后TaqMan PCR分析。茎环RT引物在RT效率和特异性方面优于常规引物。TaqMan miRNA检测对成熟的miRNA具有特异性，并可区分仅相差一个核苷酸的相关miRNA。此外，它们不受基因组DNA污染的影响。对于大多数miRNA，常规地用少至25 pg的总RNA实现精确定量。事实上，该方法的高灵敏度、特异性和精确度允许直接分析单个细胞而无需核酸纯化。与标准TaqMan基因表达测定一样，TaqMan miRNA测定显示出7个数量级的动态范围。在7个小鼠组织中的5个miRNAs的定量显示每个细胞从少于10个拷贝到超过30000个拷贝的变化。该方法能够快速、准确和灵敏地进行miRNA表达谱分析，并可以识别和监测组织或疾病特异性的潜在生物标志物。茎环RT-PCR可用于其他小RNA分子如短干扰RNA（siRNA）的定量。此外，茎环RT引物设计的概念可以应用于小RNA克隆和多重检测，以获得更好的特异性和效率。
A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
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影响因子：
56.9
作者：
Lagos-Quintana, M;Rauhut, R;Tuschl, T
通讯作者：
Tuschl, T
影响因子：
14.9
作者：
Griffiths-Jones, S
通讯作者：
Griffiths-Jones, S
影响因子：
10.5
作者：
Mourelatos, Z;Dostie, J;Dreyfuss, G
通讯作者：
Dreyfuss, G
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
影响因子：
56.9
作者：
Lim, LP;Glasner, ME;Bartel, DP
通讯作者：
Bartel, DP