Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs
Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs
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DOI：
10.1261/rna.2148705
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发表时间：
2005-11-01
期刊：
影响因子：
4.5
通讯作者：
Johnson, JM
中科院分区：
文献类型：
--
作者：
Raymond, CK;Roberts, BS;Johnson, JM
人们对microRNAs生物学和RNA干扰技术的兴趣激增。我们描述了一种简单、可靠、廉价的定量分析microRNA和短干扰RNA的方法。该方法依赖于通过反转录将RNA延伸到cDNA，然后进行实时定量聚合酶链式反应。给出了影响分析成功的关键技术参数。对微小RNA水平的测量是敏感的，大多数检测允许在毫微摩尔范围内进行测量，这相当于每个细胞数十个拷贝或更少。该分析具有高动态范围，并提供跨越6-7个数量级的microRNA浓度差异的线性读数。该分析能够区分通过细微序列差异而不同的相关microRNA家族成员。我们使用该方法对12个组织样本中的6个microRNAs进行了定量分析。这些数据证实了这些非编码调控RNA表达模式的显著变化。
There has been a surge of interest in the biology of microRNAs and the technology of RNA interference. We describe a simple, robust, inexpensive assay for quantitative analysis of microRNAs and short-interfering RNAs. The method relies on primer extension conversion of RNA to cDNA by reverse transcription followed by quantitative, real-time PCR. Technical parameters critical to the success of the assay are presented. Measurements of microRNA levels are sensitive, with most assays allowing measurements in the femtomolar range, which corresponds to tens of copies per cell or less. The assay has a high dynamic range and provides linear readout over differences in microRNA concentrations that span 6-7 orders of magnitude. The assay is capable of discriminating between related microRNA family members that differ by subtle sequence differences. We used the method for quantitative analysis of six microRNAs across 12 tissue samples. The data confirm striking variation in the patterns of expression of these noncoding regulatory RNAs.