Summary
MicroRNAs (miRNAs) are small regulatory RNAs varying in length between 20 and 24 nucleotides. They are thought to play a key role during development by negative gene regulation at the post-transcriptional level. Recent studies using quantitative polymerase chain reaction (QPCR) and northern blot analysis have reported the presence of several miRNA unique to specific cell types. The NCode™ multispecies miRNA array provides a means for simultaneously profiling the expression patterns of hundreds of known miRNAs in a given cell type or biological sample. Using this method, miRNA expression patterns in embryonic and adult stem cell lines can be characterized and compared with each other. The accuracy of NCode™ miRNA array data can be further confirmed by QPCR analysis of putative array hits. This array-based screening platform is a fast and easy to use analytical tool that allows one to asses the state of stem cell lines following multiple passages in culture as well as a discovery tool that eliminates the need to screen large numbers of candidate regulatory miRNAs by northern blot or PCR. In this chapter, we describe in detail the method to carry out miRNA array analysis in human embryonal carcinoma cells and confirm the array results using QPCR.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function (2004)) Cell, 116, 281–97.
Lee, R. C., Feinbaum, R. L., and Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 (1993) Cell, 75, 843–54.
Wightman, B., Ha, I., and Ruvkun, G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans (1993) Cell, 75, 855–62.
Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. Identification of novel genes coding for small expressed RNAs (2001) Science, 294, 853–8.
Moss, E. G. MicroRNAs: hidden in the genome (2002) Curr Biol, 12, R138–40.
Pasquinelli, A. E., Reinhart, B. J., Slack, F., Martindale, M. Q., Kuroda, M. I., Maller, B., Hayward, D. C., Ball, E. E., Degnan, B., Muller, P., Spring, J., Srinivasan, A., Fishman, M., Finnerty, J., Corbo, J., Levine, M., Leahy, P., Davidson, E., and Ruvkun, G. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA (2000) Nature, 408, 86–9.
Olsen, P. H., and Ambros, V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation (1999) Dev Biol, 216, 671–80.
Bartel, B., and Bartel, D. P. MicroRNAs: at the root of plant development? (2003) Plant Physiol, 132, 709–17.
Aravin, A. A., Lagos-Quintana, M., Yalcin, A., Zavolan, M., Marks, D., Snyder, B., Gaasterland, T., Meyer, J., and Tuschl, T. The small RNA profile during Drosophila melanogaster development (2003) Dev Cell, 5, 337–50.
Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J., Lendeckel, W., and Tuschl, T. Identification of tissue-specific microRNAs from mouse (2002) Curr Biol, 12, 735–9.
Krichevsky, A. M., King, K. S., Donahue, C. P., Khrapko, K., and Kosik, K. S. A microRNA array reveals extensive regulation of microRNAs during brain development (2003) RNA, 9, 1274–81.
Sempere, L. F., Sokol, N. S., Dubrovsky, E. B., Berger, E. M., and Ambros, V. Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and broad-complex gene activity (2003) Dev Biol, 259, 9–18.
Houbaviy, H. B., Murray, M. F., and Sharp, P. A. Embryonic stem cell-specific MicroRNAs (2003) Dev Cell, 5, 351–8.
Suh, M. R., Lee, Y., Kim, J. Y., Kim, S. K., Moon, S. H., Lee, J. Y., Cha, K. Y., Chung, H. M., Yoon, H. S., Moon, S. Y., Kim, V. N., and Kim, K. S. Human embryo- nic stem cells express a unique set of microRNAs (2004) Dev Biol, 270, 488–98.
Krichevsky, A. M., Sonntag, K. C., Isacson, O., and Kosik, K. S. Specific microRNAs modulate embryonic stem cell-derived neurogenesis (2006) Stem Cells, 24, 857–64.
Chen, C. Z., Li, L., Lodish, H. F., and Bartel, D. P. MicroRNAs modulate hematopoietic lineage differentiation (2004) Science, 303, 83–6.
Chen, J. F., Mandel, E. M., Thomson, J. M., Wu, Q., Callis, T. E., Hammond, S. M., Conlon, F. L., and Wang, D. Z. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation (2006) Nat Genet, 38, 228–33.
Yi, R., O’Carroll, D., Pasolli, H. A., Zhang, Z., Dietrich, F. S., Tarakhovsky, A., and Fuchs, E. Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs (2006) Nat Genet, 38, 356–62.
Goff, L. A., Yang, M., Bowers, J., Getts, R. C., Padgett, R. W., and Hart, R. P. Rational probe optimization and enhanced detection strategy for microRNAs using microarrays (2005) RNA Biol, 2, 93–100.
Kerr, M. K., Martin, M., and Churchill, G. A. Analysis of variance for gene expression microarray data (2000) J Comput Biol, 7, 819–37.
Kerr, M. K., and Churchill, G. A. Experimental design for gene expression microarrays (2001) Biostatistics, 2, 183–201.
Manly, B. F. J. (1997) Randomization, bootstrap and Monte Carlo Methods in Biology. Second Edition. Chapman and Hall/CRC, Boca Raton.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press
About this protocol
Cite this protocol
Lakshmipathy, U., Love, B., Adams, C., Thyagarajan, B., Chesnut, J.D. (2007). Micro RNA Profiling. In: Vemuri, M.C. (eds) Stem Cell Assays. Methods in Molecular Biology™, vol 407. Humana Press. https://doi.org/10.1007/978-1-59745-536-7_8
Download citation
DOI: https://doi.org/10.1007/978-1-59745-536-7_8
Publisher Name: Humana Press
Print ISBN: 978-1-58829-744-0
Online ISBN: 978-1-59745-536-7
eBook Packages: Springer Protocols