European Molecular Biology Laboratory, Heidelberg D 69117, Germany.
Methods. 2010 Apr;50(4):244-9. doi: 10.1016/j.ymeth.2010.01.026. Epub 2010 Jan 28.
We review different methodologies to estimate the expression levels of microRNAs (miRNAs) using real-time quantitative PCR (qPCR). As miRNA analysis is a fast changing research field, we have introduced novel technological approaches and compared them to existing qPCR profiling methodologies. qPCR also remains the method of choice for validating results obtained from whole-genome screening (e.g. with microarray). In contrast to presenting a stepwise description of different platforms, we discuss expression profiling of mature miRNAs by qPCR in four sequential sections: (1) cDNA synthesis; (2) primer design; (3) detection of amplified products; and (4) data normalization. We address technical challenges associated with each of these and outline possible solutions.
我们综述了不同的方法来使用实时定量 PCR（qPCR）来估计 microRNAs（miRNAs）的表达水平。由于 miRNA 分析是一个快速变化的研究领域，我们引入了新的技术方法，并将其与现有的 qPCR 分析方法进行了比较。qPCR 仍然是验证全基因组筛选（例如微阵列）获得的结果的首选方法。与呈现不同平台的逐步描述不同，我们通过 qPCR 按四个连续部分讨论成熟 miRNAs 的表达谱：（1）cDNA 合成；（2）引物设计；（3）扩增产物的检测；和（4）数据归一化。我们解决了与这些方法相关的技术挑战，并概述了可能的解决方案。
