Abstract
MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression controlling central biological processes. These small, approx 22-nucleotide (nt)-long RNA molecules induce translational suppression when they are imperfectly matched to their target messenger RNA (mRNA) or direct mRNA cleavage when perfectly, or nearly perfectly, matched to their target. Direct roles in developmental processes have been described in a variety of species, and involvement in human diseases, such as cancer and diabetes, has been implied. These studies highlight the need to obtain detailed expression profiles of miRNAs in tissues, during development, and in disease. Their small size and the existence of miRNA families of related sequences pose critical problems in approaching expression analysis of miRNAs, especially using high-throughput approaches. All methodologies presented here address the special requirements for the analysis of miRNA expression using a variety of platforms, including cloning, microarrays, and microbeads. The different variables, as well as the different approaches, used by various laboratories are detailed and general recommendations are provided.
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References
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.
Lee, R. C., Feinbaum, R. L., and Ambros V. (1993) The C. elegans heterochronic genelin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.
Reinhart, B. J., Slack, F. J., Basson, H., et al. (2000) The 21-nucleotide let-7 RNA regulatesdevelopmental timing in Caenorhabditis elegans. Nature 403, 901–906.
Pasquinelli, A. E., Reinhart, B. J., Slack, F., et al. (2000) Conservation of the sequenceand temporal expression of let-7 heterochronic regulatory RNA. Nature 408, 86–89.
Berezikov, E., Guryev, V., van de Belt, J., Weinholds, E., Plasterk, R. H., and Cuppen, E.(2005) Phylogenetic shadowing and computational identification of human microRNAgenes. Cell 120, 21–24.
**e, X., Lu, J., Kulbokas, E. J., et al. (2005) Systematic discovery of regulatory motifs inhuman promoters and 3′ UTRs by comparison of several mammals. Nature 434, 338–345.
Bentwich, I., Avniel, A., Karov, Y., et al. (2005) Identification of hundreds of conservedand nonconserved human microRNAs. Nat. Genet. 37, 766–770.
Carrington, J. C. and Ambros, V. (2003) Role of microRNAs in plant and animal development. Science 301, 336–338.
Bartel, B. and Bartel, D. P. (2003) MicroRNAs: at the root of plant development? PlantPhysiol. 132, 709–717.
Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116,281–297.
Ambros, V. (2004) The functions of animal microRNAs. Nature 431, 350–355.
Bartel, D. P. and Chen, C. Z. (2004) Micromanagers of gene expression: the potentiallywidespread influence of metazoan microRNAs. Nat. Rev. Genet. 5, 396–400.
Hobert, O. (2004) Common logic of transcription factor and microRNA action. Trends Biochem.Sci. 29, 462–468.
Murchison, E. P. and Hannon, G. J. (2004) miRNAs on the move: miRNA biogenesis andthe RNAi machinery. Curr. Opin. Cell Biol. 16, 223–229.
Mattick, J. S. and Makunin, I. V. (2005) Small regulatory RNAs in mammals. Hum. Mol.Genet. 14(Spec 1), R121–R132.
Bentwich, I. (2005) A postulated role for microRNA in cellular differentiation. FASEB J.In press.
Kim, V. N. (2005) MicroRNA biogenesis: coordinated crop** and dicing. Nat. Rev. Mol.Cell Biol. 6, 376–385.
Lee, Y., Jeon, K., Lee, J. T., Kim, S., and Kim, V. N. (2002) MicroRNA maturation:stepwise processing and subcellular localization. EMBO J. 21, 4663–4670.
Lee, Y., Ahn, C., Han, J., et al. (2003) The nuclear RNase III Drosha initiates microRNAprocessing. Nature 425, 415–419.
Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F., and Hannon, G. J. (2004) Processingof primary microRNAs by the Microprocessor complex. Nature 432, 231–235.
Gregory, R. I., Yan, K. P., Amuthan, G., et al. (2004) The Microprocessor complex mediatesthe genesis of microRNAs. Nature 432, 235–240.
Ketting, R. F., Fischer, S. E., Bernstein, E., Sijen, T., Hannon, G. J., and Plasterk, R.(2001) Dicer functions in RNA interference and in synthesis of small RNA involved indevelopmental timing in C. elegans. Genes Dev. 15, 2654–2659.
Hutvagner, G. and Zamore, P. D. (2002) A microRNA in a multiple-turnover RNAi enzymecomplex. Science 297, 2056–2060.
Hake, S. (2003) MicroRNAs: a role in plant development. Curr. Biol. 13, R851–R852.
Yekta, S., Shih, I. H., and Bartel, D. P. (2004) MicroRNA-directed cleavage of HOXB8mRNA. Science 304, 594–596.
Lim, L. P., Lau, N. C., Garrett-Engele, P., et al. (2005) Microarray analysis shows thatsome microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773.
Esau, C., Kang, X., Peralta, E., et al. (2004) MicroRNA-143 regulates adipocyte differentiation. J. Biol. Chem. 279, 52,361–52,365.
Poy, M. N., Eliasson, L., Krutzfeldt, J., et al. (2004) A pancreatic islet-specific microRNAregulates insulin secretion. Nature 432, 226–230.
Calin, G. A., Dumitru, C. D., Shimizu, M., et al. (2002) Frequent deletions and downregulationof micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 99, 15,524–15,529.
Michael, M. Z., O’Connor, S. M., van Holst Pellekaan N. G., Young G. P., and James R. J. (2003) Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol.Cancer Res. 1, 882–891.
Calin, G. A., Liu, C. G., Sevignani, C., et al. (2004) MicroRNA profiling reveals distinctsignatures in B cell chronic lymphocytic leukemias. Proc. Natl. Acad. Sci. USA 101, 11,755–11,760.
Takamizawa, J., Konishi, H., Yanagisawa, K., et al. (2004) Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 64, 3753–3756.
Calin, G. A., Sevignani, C., Dumitru, C. D., et al. (2004) Human microRNA genes are frequentlylocated at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad.Sci. USA 101 (9), 2999–3004.
Metzler, M., Wilda, M., Busch, K., Viehmann, S., and Borkhardt, A. (2004) High expressionof precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes ChromosomesCancer 39, 167–169.
He, L., Thomson, J. M., Hemann, M. T., et al. (2005) A microRNA polycistron as a potentialhuman oncogene. Nature 435, 828–833.
Lu, J., Getz, G., Miska, E. A., et al. (2005) MicroRNA expression profiles classify humancancers. Nature 435, 834–838.
O’Donnell, K. A., Wentzel, E. A., Zeller, K. I., Dang, C. V., and Mendell, J. T. (2005) c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839–843.
Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001) Identification ofnovel genes coding for small expressed RNAs. Science 294, 853–858.
Lau, N. C., Lim, L. P., Weinstein, E. G., and Bartel, D. P. (2001) An abundant class of tinyRNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862.
Lee, R. C. and Ambros, V. (2001) An extensive class of small RNAs in Caenorhabditiselegans. Science 294, 862–864.
Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J., Lendeckel, W., and Tuschl, T.(2002) Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739.
Lagos-Quintana, M., Rauhut, R., Meyer, J., Borkhardt, A., and Tuschl, T. (2003) NewmicroRNAs from mouse and human. RNA 9, 175–179.
Chen, P. Y., Manninga, H., Slanchev, K., et al. (2005) The developmental miRNA profilesof zebrafish as determined by small RNA cloning. Genes Dev. 19, 1288–1293.
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, 1274–1281.
Barad, O., Meiri, E., Avniel, A., et al. (2004) MicroRNA expression detected by oligonucleotidemicroarrays: System establishment and expression profiling in human tissues. Genome Res. 14, 2486–2494.
Liu, C. G., Calin, G. A., Meloon, B., et al. (2004) An oligonucleotide microchip for genomewidemicroRNA profiling in human and mouse tissues. Proc. Natl. Acad. Sci. USA 101,9740–9744.
Thomson, J. M., Parker, J., Perou, C. M., and Hammond, S. M. (2004) A custom microarrayplatform for analysis of microRNA gene expression. Nat. Methods 1, 47–53.
Babak, T., Zhang, W., Morris, Q., Blencowe, B. J., and Hughes, T. R. (2004) ProbingmicroRNAs with microarrays: tissue specificity and functional inference. RNA 10, 1813–1819.
Peng, W. T., Robinson, M. D., Mnaimneh, S., et al. (2003) A panoramic view of yeastnoncoding RNA processing. Cell 113, 919–933.
Sempere, L. F., Freemantle, S., Pitha-Rowe, I., Moss, E., Dmitrovsky, E. and Ambros, V.(2004) Expression profiling of mammalian microRNAs uncovers a subset of brain-expressedmicroRNAs with possible roles in murine and human neuronal differentiation. Genome Biol. 5, R13.
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 ofmicroRNAs. Nat Methods 1, 155–161.
Liang, R. Q., Li, W., Li, Y., et al. (2005) An oligonucleotide microarray for microRNAexpression analysis based on labeling RNA with quantum dot and nanogold probe. NucleicAcids Res. 33, e17.
Miska, E. A., Alvarez-Saavedra, E., Townsend, M. et al. (2004) Microarray analysis ofmicroRNA expression in the develo** mammalian brain. Genome Biol. 5, R68.
Baskerville, S. and Bartel, D. P. (2005) Microarray profiling of microRNAs reveals frequentcoexpression with neighboring miRNAs and host genes. RNA 11, 241–247.
Williams, K. P. and Bartel, D. P. (1995) PCR product with strands of unequal length. NucleicAcids Res. 23, 4220–4221.
Hartig, J. S., Grune I., Najafi-Shoushtarai S. H., and Famulok M. (2004) Sequence-specificdetection of MicroRNAs by signal-amplifying ribozymes. J. Am. Chem. Soc. 126,722–723.
Brenner, S., Johnson M., Bridgham J., et al. (2000) Gene expression analysis by massivelyparallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol. 18, 630–634.
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Einat, P. (2006). Methodologies for High-Throughput Expression Profiling of MicroRNAs. In: Ying, SY. (eds) MicroRNA Protocols. Methods in Molecular Biology™, vol 342. Humana Press. https://doi.org/10.1385/1-59745-123-1:139
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DOI: https://doi.org/10.1385/1-59745-123-1:139
Publisher Name: Humana Press
Print ISBN: 978-1-58829-581-1
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