Kang Hara, Hata Akiko
Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, 406-772, Republic of Korea.
Cardiovascular Research Institute, University of California, 555 Mission Bay Blvd. South, Rm. 252T, San Francisco, CA, 94158-9001, USA.
Methods Mol Biol. 2016;1344:313-23. doi: 10.1007/978-1-4939-2966-5_20.
The signaling pathway of TGF-β and its family member BMP has been implicated in vascular development and maintenance of homeostasis by modulating expression of small noncoding microRNAs (miRNAs). MiRNAs repress target genes, which play a critical role in regulating vascular smooth muscle cell (VSMC) growth, phenotype, and function. To understand the mechanisms by which specific miRNAs control the TGF-β and BMP signaling pathway in VSMC, it is essential to quantitate levels of specific miRNAs and their precursors whose expression are controlled by TGF-β/BMP signaling. Here, we describe a real-time quantization method for accurate and sensitive detection of miRNAs and their precursors, such as primary transcripts of miRNAs (pri-miRNAs) and precursor miRNAs (pre-miRNAs). This method requires two steps; synthesis of single-stranded complementary DNAs (cDNAs) from total RNA samples and quantization of specific pri-, pre-, or mature miRNAs by quantitative polymerase chain reaction (PCR) using a real-time PCR machine.
转化生长因子-β（TGF-β）及其家族成员骨形态发生蛋白（BMP）的信号通路，通过调节小型非编码微小RNA（miRNA）的表达，参与了血管发育和体内稳态的维持。miRNA会抑制靶基因，而靶基因在调节血管平滑肌细胞（VSMC）的生长、表型和功能方面起着关键作用。为了了解特定miRNA控制VSMC中TGF-β和BMP信号通路的机制，对其表达受TGF-β/BMP信号控制的特定miRNA及其前体的水平进行定量至关重要。在此，我们描述了一种用于准确、灵敏地检测miRNA及其前体（如miRNA的初级转录本（pri-miRNA）和前体miRNA（pre-miRNA））的实时定量方法。该方法需要两个步骤：从总RNA样本中合成单链互补DNA（cDNA），以及使用实时PCR仪通过定量聚合酶链反应（PCR）对特定的pri-、pre-或成熟miRNA进行定量。
