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Quantitation of MicroRNAs by Real-Time RT-qPCR

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PCR Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 687))

Abstract

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.

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References

  1. Lee, R.C., Feinbaum, R.L., and Ambros, V. (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–54.

    Article  PubMed  CAS  Google Scholar 

  2. Wightman, B., Ha, I., and Ruvkun, G. (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75, 855–62.

    Article  PubMed  CAS  Google Scholar 

  3. Ruvkun, G. (2001) Molecular biology. Glimpses of a tiny RNA world. Science 294, 797–9.

    Article  PubMed  CAS  Google Scholar 

  4. Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–97.

    Article  PubMed  CAS  Google Scholar 

  5. Griffiths-Jones, S., Saini, H.K., van Dongen, S., and Enright, A.J. (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36, D154–8.

    Article  PubMed  CAS  Google Scholar 

  6. Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science 294, 853–8.

    Article  PubMed  CAS  Google Scholar 

  7. Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–62.

    Article  PubMed  CAS  Google Scholar 

  8. Pillai, R.S., Bhattacharyya, S.N., Artus, C.G., Zoller, T., Cougot, N., Basyuk, E., Bertrand, E., and Filipowicz, W. (2005) Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309, 1573–6.

    Article  PubMed  CAS  Google Scholar 

  9. Orom, U.A., Nielsen, F.C., and Lund, A.H. (2008) MicroRNA-10a binds the 5′-UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell 30, 460–71.

    Article  PubMed  Google Scholar 

  10. Lewis, B.P., Burge, C.B., and Bartel, D.P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20.

    Article  PubMed  CAS  Google Scholar 

  11. Brennecke, J., Stark, A., Russell, R.B., and Cohen, S.M. (2005) Principles of microRNA/target recognition. PLoS Biol 3, e85.

    Article  PubMed  Google Scholar 

  12. Xie, X., Lu, J., Kulbokas, E.J., Golub, T.R., Mootha, V., Lindblad-Toh, K., Lander, E.S., and Kellis, M. (2005) Systematic discovery of regulatory motifs in human promoters and 3′UTRs by comparison of several mammals. Nature 434, 338–45.

    Article  PubMed  CAS  Google Scholar 

  13. Lim, L.P., Lau, N.C., Garrett-Engele, P., Grimson, A., Schelter, J.M., Castle, J., Bartel, D.P., Linsley, P.S., and Johnson, J.M. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–73.

    Article  PubMed  CAS  Google Scholar 

  14. Giraldez, A.J., Mishima, Y., Rihel, J., Grocock, R.J., Van Dongen, S., Inoue, K., Enright, A.J., and Schier, A.F. (2006) Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–9.

    Article  PubMed  CAS  Google Scholar 

  15. Makeyev, E.V. and Maniatis, T. (2008) Multilevel regulation of gene expression by microRNAs. Science 319, 1789–90.

    Article  PubMed  CAS  Google Scholar 

  16. Zhao, Y. and Srivastava, D. (2007) A developmental view of microRNA function. Trends Biochem Sci 32, 189–97.

    Article  PubMed  CAS  Google Scholar 

  17. Visvanathan, J., Lee, S., Lee, B., Lee, J.W., and Lee, S.K. (2007) The microRNA miR-124 antagonizes the ... pathway during embryonic CNS development. Genes Dev 21, 744–9.

    Article  PubMed  CAS  Google Scholar 

  18. Conaco, C., Otto, S., Han, J.J., and Mandel, G. (2006) Reciprocal actions of REST and a microRNA promote neuronal identity. Proc Natl Acad Sci USA 103, 2422–7.

    Article  PubMed  CAS  Google Scholar 

  19. Makeyev, E.V., Zhang, J., Carrasco, M.A., and Maniatis, T. (2007) The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol Cell 27, 435–48.

    Article  PubMed  CAS  Google Scholar 

  20. Boutz, P.L., Chawla, G., Stoilov, P., and Black, D.L. (2007) MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development. Genes Dev 21, 71–84.

    Article  PubMed  CAS  Google Scholar 

  21. Edwards, R.H., Marquitz, A.R., and Raab-Traub, N. (2008) Epstein-Barr virus BART miRNAs are produced from a large intron prior to splicing. J Virol 82, 9094–106.

    Article  PubMed  CAS  Google Scholar 

  22. Zhang, R., Wang, Y.Q., and Su, B. (2008) Molecular evolution of a primate-specific microRNA family. Mol Biol Evol 25, 1493–502.

    Article  PubMed  CAS  Google Scholar 

  23. Altuvia, Y., Landgraf, P., Lithwick, G., Elefant, N., Pfeffer, S., Aravin, A., Brownstein, M.J., Tuschl, T., and Margalit, H. (2005) Clustering and conservation patterns of human microRNAs. Nucleic Acids Res 33, 2697–706.

    Article  PubMed  CAS  Google Scholar 

  24. Gaur, A., Jewell, D.A., Liang, Y., Ridzon, D., Moore, J.H., Chen, C., Ambros, V.R., and Israel, M.A. (2007) Characterization of microRNA expression levels and their biological correlates in human cancer cell lines. Cancer Res 67, 2456–68.

    Article  PubMed  CAS  Google Scholar 

  25. Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A., Pfeffer, S., Rice, A., Kamphorst, A.O., Landthaler, M., et al. (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–14.

    Article  PubMed  CAS  Google Scholar 

  26. Rosenfeld, N., Aharonov, R., Meiri, E., Rosenwald, S., Spector, Y., Zepeniuk, M., Benjamin, H., Shabes, N., Tabak, S., Levy, A., et al. (2008) MicroRNAs accurately identify cancer tissue origin. Nat Biotechnol 26, 462–9.

    Article  PubMed  CAS  Google Scholar 

  27. Chen, C., Ridzon, D., Lee, C.T., Blake, J., Sun, Y., and Strauss, W.M. (2007) Defining embryonic stem cell identity using differentiation-related microRNAs and their potential targets. Mamm Genome 18, 316–27.

    Article  PubMed  CAS  Google Scholar 

  28. Xi, Y., Nakajima, G., Gavin, E., Morris, C.G., Kudo, K., Hayashi, K., and Ju, J. (2007) Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA 13, 1668–74.

    Article  PubMed  CAS  Google Scholar 

  29. Mitchell, P.S., Parkin, R.K., Kroh, E.M., Fritz, B.R., Wyman, S.K., Pogosova-Agadjanyan, E.L., Peterson, A., Noteboom, J., O’Briant, K.C., Allen, A., et al. (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105, 10513–18.

    Article  PubMed  CAS  Google Scholar 

  30. Chen, C., Ridzon, D.A., Broomer, A.J., Zhou, Z., Lee, D.H., Nguyen, J.T., Barbisin, M., Xu, N.L., Mahuvakar, V.R., Andersen, M.R., et al. (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33, e179.

    Article  PubMed  Google Scholar 

  31. Gilad, S., Meiri, E., Yogev, Y., Benjamin, S., Lebanony, D., Yerushalmi, N., Benjamin, H., Kushnir, M., Cholakh, H., Melamed, N., et al. (2008) Serum microRNAs are promising novel biomarkers. PLoS One 3, e3148.

    Article  PubMed  Google Scholar 

  32. Lu, J., Getz, G., Miska, E.A., Alvarez-Saavedra, E., Lamb, J., Peck, D., Sweet-Cordero, A., Ebert, B.L., Mak, R.H., Ferrando, A.A., et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435, 834–8.

    Article  PubMed  CAS  Google Scholar 

  33. Calin, G.A., Liu, C.G., Sevignani, C., Ferracin, M., Felli, N., Dumitru, C.D., Shimizu, M., Cimmino, A., Zupo, S., Dono, M., et al. (2004) MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci USA 101, 11755–60.

    Article  PubMed  CAS  Google Scholar 

  34. Krichevsky, A.M., King, K.S., Donahue, C.P., Khrapko, K., and Kosik, K.S. (2003) A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9, 1274–81.

    Article  PubMed  CAS  Google Scholar 

  35. Nelson, P.T., Baldwin, D.A., Scearce, L.M., Oberholtzer, J.C., Tobias, J.W., and Mourelatos, Z. (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1, 155–61.

    Article  PubMed  CAS  Google Scholar 

  36. Sarkar, D., Parkin, R., Wyman, S., Bendoraite, A., Sather, C., Delrow, J., Godwin, A.K., Drescher, C., Huber, W., Gentleman, R., et al. (2008) Quality assessment and data analysis for microRNA expression arrays. Nucleic Acids Res 37, e17.

    Article  PubMed  Google Scholar 

  37. Raymond, C.K., Roberts, B.S., Garrett-Engele, P., Lim, L.P., and Johnson, J.M. (2005) Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737–44.

    Article  PubMed  CAS  Google Scholar 

  38. Shi, R. and Chiang, V.L. (2005) Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 39, 519–25.

    Article  PubMed  CAS  Google Scholar 

  39. Sharbati-Tehrani, S., Kutz-Lohroff, B., Bergbauer, R., Scholven, J., and Einspanier, R. (2008) miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample. BMC Mol Biol 9, 34.

    Article  PubMed  Google Scholar 

  40. Chen, J., Lozach, J., Garcia, E.W., Barnes, B., Luo, S., Mikoulitch, I., Zhou, L., Schroth, G., and Fan, J.B. (2008) Highly sensitive and specific microRNA expression profiling using BeadArray technology. Nucleic Acids Res 36, e87.

    Article  PubMed  Google Scholar 

  41. Allawi, H.T., Dahlberg, J.E., Olson, S., Lund, E., Olson, M., Ma, W.-P., Takova, T., Neri, B.P., and Lyamichev, V.I. (2004) Quantitation of microRNAs using a modified Invader assay. RNA 10, 1153–61.

    Article  PubMed  CAS  Google Scholar 

  42. Jonstrup, S.P., Koch, J., and Kjems, J. (2006) A microRNA detection system based on padlock probes and rolling circle amplification. RNA 12, 1747–52.

    Article  PubMed  CAS  Google Scholar 

  43. Schmittgen, T.D., Lee, E.J., Jiang, J., Sarkar, A., Yang, L., Elton, T.S., and Chen, C. (2008) Real-time PCR quantification of precursor and mature microRNA. Methods 44, 31–8.

    Article  PubMed  CAS  Google Scholar 

  44. Mestdagh, P., Feys, T., Bernard, N., Guenther, S., Chen, C., Speleman, F., and Vandesompele, J. (2008) High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. Nucleic Acids Res 36, e143.

    Article  PubMed  Google Scholar 

  45. de Kok, J.B., Roelofs, R.W., Giesendorf, B.A., Pennings, J.L., Waas, E.T., Feuth, T., Swinkels, D.W., and Span, P.N. (2005) Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest 85, 154–9.

    PubMed  Google Scholar 

  46. Suzuki, T., Higgins, P.J., and Crawford, D.R. (2000) Control selection for RNA quantitation. Biotechniques 29, 332–7.

    PubMed  CAS  Google Scholar 

  47. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, RESEARCH0034.

    Article  PubMed  Google Scholar 

  48. Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95, 14863–8.

    Article  PubMed  CAS  Google Scholar 

  49. Andersen, C.L., Jensen, J.L., and Orntoft, T.F. (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64, 5245–50.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Dr. Neil Straus for comments and feedback on the manuscript.

Author information

Authors and Affiliations

  1. Genomic Assays R&D, Molecular Biology Systems Division, Life Technologies, Foster City, CA, USA

    Caifu Chen & Jason Halsey

  2. Molecular Biology Systems Division, Life Technologies, Foster City, CA, USA

    Ruoying Tan, Linda Wong & Richard Fekete

Authors
  1. Caifu Chen
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  2. Ruoying Tan
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  3. Linda Wong
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  4. Richard Fekete
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  5. Jason Halsey
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Corresponding author

Correspondence to Caifu Chen .

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Editors and Affiliations

  1. , Department of Pathology, The University of Melbourne, Corner of Grattan St & Royal Parade, Parkville, 3010, Victoria, Australia

    Daniel J. Park

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© 2011 Humana Press

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Chen, C., Tan, R., Wong, L., Fekete, R., Halsey, J. (2011). Quantitation of MicroRNAs by Real-Time RT-qPCR. In: Park, D. (eds) PCR Protocols. Methods in Molecular Biology, vol 687. Humana Press. https://doi.org/10.1007/978-1-60761-944-4_8

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  • DOI: https://doi.org/10.1007/978-1-60761-944-4_8

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-943-7

  • Online ISBN: 978-1-60761-944-4

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