title: Quantitative Real-Time PCR Analysis of MicroRNAs and Their Precursors Regulated by TGF-β Signaling.
abstract: 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.
authors.affiliation: 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.
authors.forename: Hara
authors.initials: H
authors.lastname: Kang
authors.affiliation: Cardiovascular Research Institute, University of California, 555 Mission Bay Blvd. South, Rm. 252T, San Francisco, CA, 94158-9001, USA. akiko.hata@ucsf.edu.
authors.forename: Akiko
authors.initials: A
authors.lastname: Hata
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D035683:MicroRNAs; D012322:RNA Precursors; D016212:Transforming Growth Factor beta
keywords: BMP; MicroRNA; Quantitative real-time PCR; TGF-β