Chen Caifu, Tan Ruoying, Wong Linda, Fekete Richard, Halsey Jason
Genomic Assays R&D, Molecular Biology Systems Division, Life Technologies, Foster City, CA, USA.
Methods Mol Biol. 2011;687:113-34. doi: 10.1007/978-1-60761-944-4_8.
MicroRNAs (miRNAs) are ∼22 nucleotide regulatory RNA molecules that play important roles in controlling developmental and physiological processes in animals and plants. Measuring the level of miRNA expression is a critical step in methods that study the regulation of biological functions and that use miRNA profiles as diagnostic markers for cancer and other diseases. Even though the quantitation of these small miRNA molecules by RT-qPCR is challenging because of their short length and sequence similarity, a number of quantitative RT-qPCR-based miRNA quantitation methods have been introduced since 2004. The most commonly used methods are stem-loop reverse transcription (RT)-based TaqMan(®) MicroRNA assays and arrays. The high sensitivity and specificity, large dynamic range, and simple work flow of TaqMan(®) MicroRNA assays and arrays have made TaqMan analysis the method of choice for miRNA expression profiling and follow-up validation. Other methods such as poly (A) tailing-based and direct RT-based SYBR miRNA assays are also discussed in this chapter.
微小RNA（miRNA）是约22个核苷酸的调控RNA分子，在控制动植物的发育和生理过程中发挥重要作用。测量miRNA表达水平是研究生物学功能调控以及将miRNA谱用作癌症和其他疾病诊断标志物的方法中的关键步骤。尽管由于这些小miRNA分子长度短且序列相似，通过逆转录定量聚合酶链反应（RT-qPCR）对其进行定量具有挑战性，但自2004年以来已引入了许多基于RT-qPCR的miRNA定量方法。最常用的方法是基于茎环逆转录（RT）的TaqMan®微小RNA检测法和芯片法。TaqMan®微小RNA检测法和芯片法具有高灵敏度和特异性、大动态范围以及简单的工作流程，这使得TaqMan分析成为miRNA表达谱分析和后续验证的首选方法。本章还讨论了其他方法，如基于聚（A）加尾和基于直接RT的SYBR miRNA检测法。
