Abstract
MicroRNAs (miRNAs) are ubiquitous regulators of gene expression in plants and ani-mals. Their distinctive structure, as very short RNAs with a 5′-phosphate and 3′-hydroxyl group, has enabled the development of protocols to clone miRNAs. After enrichment of these small molecules by size, serial ligation of adapter oligonucleotides to each terminus allows amplification using reverse transcription (RT)-polymerase chain reaction (PCR). Plasmid cloning of multiple miRNA sequences and subsequent DNA sequence analysis enable both bioinformatic characterization of the various miRNAs and experimental vali-dation of their accumulation in cells.
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
Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349.
Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F., and Hannon, G. J. (2004) Pro-cessing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235.
Gregory, R. I., Yan, K. P., Amuthan, G., et al. (2004) The Microprocessor complex medi-ates the genesis of microRNAs. Nature 432, 235–240.
Bannerjee, D. and Slack, F. (2002) Control of developmental timing by small temporal RNAs: a paradigm for RNA-mediated regulation of gene expression. Bioessays 24, 119–129.
Ambros, V. (2004) The functions of animal microRNAs. Nature 431, 350–355.
Bartel, D. (2004) MicroRNAs: genomics, biogenesis, mechanism and function. Cell 116, 281–297.
Poy, M. N., Eliasson, L., Krutzfeldt, J., et al. (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432, 226–230.
Pfeffer, S., Zavolan, M., Grasser, F. A., et al. (2004) Identification of virus-encoded micro-RNAs. Science 304, 734–736.
Calin, G. A., Sevignani, C., Dumitru, C. D., et al. (2004) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. USA 101, 2999–3004.
Calin, G. A., Liu, C. G., Sevignani, C., et al. (2004) MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc. Natl. Acad. Sci. USA 101, 11,755–11,760.
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.
Eis, P. S., Tam, W., Sun, L., et al. (2005) Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc. Natl. Acad. Sci. USA 102, 3627–3632.
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.
Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001) RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15, 188–200.
Pfeffer, S., Lagos-Quintana, M., and Tuschl, T. (2003) Cloning of small RNA molecules. In: Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., et al., eds.), Wiley New York, pp. 26.4.1–26.4.18.
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, 858–862.
Zucker,, M. (2003) Mfold web server for nucleic acid folding and hybridization predic-tion. Nucl. Acids Res. 31, 3406–3415.
Ambros, V., Bartel, B., Bartel, D. P., et al. (2003) A uniform system for microRNA anno-tation. RNA 9, 277–279.
Griffiths-Jones S. (2004) The microRNA registry. Nucl. Acids Res. 32, D109–D111.
Ambros, V. and Lee, R. C. (2004) Identification of microRNAs and other tiny noncoding RNAs by cDNA cloning. In: Methods in Molecular Biology, vol. 265, RNA Interference, Editing, and Modification (Gott, J. M., ed.), Humana, Totowa NJ, pp. 131–158.
Chomczynski, P. and Saachi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15, 532–534, 536-537.
Milligan, J. F. and Uhlenbeck, O. C. (1989) Synthesis of small RNAs using T7 RNA poly-merase. In: Methods in Enzymology, vol. 180, RNA Processing Part A (Dahlberg J., Abelson J., and Simon, M., eds..), Academic New York, pp 51–62.
Schmittgen, T. D., Jiang, J., Liu, Q., and Yang, L. (2004) A high-throughput method to monitor the expression of microRNA precursors. Nucl. Acids Res. 32, e43.
Liu, C.-G., Calin, G. A., Meloon, B., et al. (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc. Natl. Acad. Sci USA 101, 9740–9744.
Barad, O., Meiri, E., Avniel, A., et al. (2004) MicroRNA expression detected by oligo-nucleotide microarrays: system establishment and expression profiling in human tissues. Genome Res. 14, 2486–2494.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Michael, M.Z. (2006). Cloning MicroRNAs From Mammalian Tissues. In: Ying, SY. (eds) MicroRNA Protocols. Methods in Molecular Biology™, vol 342. Humana Press. https://doi.org/10.1385/1-59745-123-1:189
Download citation
DOI: https://doi.org/10.1385/1-59745-123-1:189
Publisher Name: Humana Press
Print ISBN: 978-1-58829-581-1
Online ISBN: 978-1-59745-123-9
eBook Packages: Springer Protocols