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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Massague J (2012) TGFbeta signalling in context. Nat Rev Mol Cell Biol 13:616–630
ten Dijke P, Arthur HM (2007) Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol 8:857–869
Davis BN, Hilyard AC, Lagna G, Hata A (2008) SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454:56–61
Hata A. Functions of microRNAs in cardiovascular biology and disease. Annu Rev Physiol 75:69–93
Kang H, Hata A. (2012) MicroRNA regulation of smooth muscle gene expression and phenotype. Curr Opin Hematol 19:224–231
Davis BN, Hilyard AC, Nguyen PH, Lagna G, Hata A. (2010) Smad proteins bind a conserved RNA sequence to promote microRNA maturation by Drosha. Mol Cell 39:373–384
Kang H, Davis-Dusenbery BN, Nguyen PH, Lal A, Lieberman J et al. (2012) Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins. J Biol Chem 287:3976–3986
Borchert GM, Lanier W, Davidson BL (2006) RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol 13:1097–1101
Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary microRNAs by the microprocessor complex. Nature 432:231–235
Han J, Lee Y, Yeom KH, Nam JW, Heo I et al (2006) Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125:887–901
Hutvagner G, McLachlan J, Pasquinelli AE, Balint E, Tuschl T et al (2001) A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293:834–838
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233
Piriyapongsa J, Jordan IK, Conley AB, Ronan T, Smalheiser NR (2011) Transcription factor binding sites are highly enriched within microRNA precursor sequences. Biol Direct 6:61
Valoczi A, Hornyik C, Varga N, Burgyan J, Kauppinen S et al (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32, e175
Fichtlscherer S, De Rosa S, Fox H, Schwietz T, Fischer A et al. (2010) Circulating microRNAs in patients with coronary artery disease. Circ Res 107:677–684
Wu Q, Lu Z, Li H, Lu J, Guo L et al (2011) Next-generation sequencing of microRNAs for breast cancer detection. J Biomed Biotechnol 2011: 597145
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33, e179
Benes V, Castoldi M. (2010) Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50:244–249
Kang H, Louie J, Weisman A, Sheu-Gruttadauria J, Davis-Dusenbery BN et al. (2012) Inhibition of microRNA-302 (miR-302) by bone morphogenetic protein 4 (BMP4) facilitates the BMP signaling pathway. J Biol Chem 287:38656–38664
Acknowledgement
We thank members of the Hata lab in particular Matt Blahna for critical reading of the manuscript. This work was supported by grants from the National Institute of Health: HL093154 and HL108317, the American Heart Association: 0940095N and the LeDucq foundation Transatlantic network grant to A.H. and the National Research Foundation of Korea (Basic Science Research Program; 2012R1A1A1042812) to H.K.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kang, H., Hata, A. (2016). Quantitative Real-Time PCR Analysis of MicroRNAs and Their Precursors Regulated by TGF-β Signaling. In: Feng, XH., Xu, P., Lin, X. (eds) TGF-β Signaling. Methods in Molecular Biology, vol 1344. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2966-5_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2966-5_20
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2965-8
Online ISBN: 978-1-4939-2966-5
eBook Packages: Springer Protocols