Quantitative Real-Time PCR Analysis of MicroRNAs and Their Precursors Regulated by TGF-β Signaling

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1344)

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.

Key words

MicroRNA TGF-β BMP Quantitative real-time PCR 
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Notes

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.

References

  1. 1.
    Massague J (2012) TGFbeta signalling in context. Nat Rev Mol Cell Biol 13:616–630PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    ten Dijke P, Arthur HM (2007) Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol 8:857–869CrossRefPubMedGoogle Scholar
  3. 3.
    Davis BN, Hilyard AC, Lagna G, Hata A (2008) SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454:56–61PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Hata A. Functions of microRNAs in cardiovascular biology and disease. Annu Rev Physiol 75:69–93Google Scholar
  5. 5.
    Kang H, Hata A. (2012) MicroRNA regulation of smooth muscle gene expression and phenotype. Curr Opin Hematol 19:224–231Google Scholar
  6. 6.
    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 39:373–384Google Scholar
  7. 7.
    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 287:3976–3986Google Scholar
  8. 8.
    Borchert GM, Lanier W, Davidson BL (2006) RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol 13:1097–1101CrossRefPubMedGoogle Scholar
  9. 9.
    Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primary microRNAs by the microprocessor complex. Nature 432:231–235CrossRefPubMedGoogle Scholar
  10. 10.
    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 125:887–901CrossRefPubMedGoogle Scholar
  11. 11.
    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 293:834–838CrossRefPubMedGoogle Scholar
  12. 12.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Piriyapongsa J, Jordan IK, Conley AB, Ronan T, Smalheiser NR (2011) Transcription factor binding sites are highly enriched within microRNA precursor sequences. Biol Direct 6:61PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    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, e175PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Fichtlscherer S, De Rosa S, Fox H, Schwietz T, Fischer A et al. (2010) Circulating microRNAs in patients with coronary artery disease. Circ Res 107:677–684Google Scholar
  16. 16.
    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: 597145Google Scholar
  17. 17.
    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, e179PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Benes V, Castoldi M. (2010) Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50:244–249Google Scholar
  19. 19.
    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 287:38656–38664Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Life Sciences, College of Life Sciences and BioengineeringIncheon National UniversityIncheonRepublic of Korea
  2. 2.Cardiovascular Research InstituteUniversity of CaliforniaSan FranciscoUSA

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