Abstract
MicroRNAs (miRNAs) are a class of small RNAs (sRNAs) of approximately 22 nucleotides in length that control eukaryotic gene expression at the translational level. They regulate a wide variety of biological processes, namely developmental timing, cell differentiation, cell proliferation, the immune response, and infection. Their identification is essential to understand eukaryotic biology. Their small size, low abundance, and high instability complicated early identification, however new generation genome sequencing approaches, such as the Roche 454 Pyrosequencer, allow for both miRNA identification and for generating miRNA profiles in a given sample. This technique avoids cloning steps in bacteria and is a fast and bias-minimized tool to discover novel miRNAs and other sRNAs on a genome-wide scale. Prior to sequencing, cDNA libraries are built for each sample using total RNA as starter material. Each cDNA library can be tagged with specific identifier sequences that allow sequencing different samples in the same chip run. Here, we describe the protocols for the construction of sRNA cDNA libraries for 454 sequencing, and we include tips for overcoming problems often encountered during cDNA library preparation.
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References
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.
Berezikov, E., Thuemmler, F., van Laake, L. W., Kondova, I., Bontrop, R., Cuppen, E., et al. (2006) Diversity of microRNAs in human and chimpanzee brain. Nat Genet 38, 1375–7.
Ciaudo, C., Servant, N., Cognat, V., Sarazin, A., Kieffer, E., Viville, S., et al. (2009) Highly dynamic and sex-specific expression of microRNAs during early ES cell differentiation. PLoS Genet 5, e1000620.
Klevebring, D., Street, N. R., Fahlgren, N., Kasschau, K. D., Carrington, J. C., Lundeberg, J., et al. (2009) Genome-wide profiling of populus small RNAs. BMC Genomics 10, 620.
Ruby, J. G., Jan, C., Player, C., Axtell, M. J., Lee, W., Nusbaum, C., et al. (2006) Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell 127, 1193–1207.
Soares, A. R., Pereira, P. M., Santos, B., Egas, C., Gomes, A. C., Arrais, J., et al. (2009) Parallel DNA pyrosequencing unveils new zebrafish microRNAs. BMC Genomics 10, 195.
Griffiths-Jones, S., Saini, H. K., van, D. S., and Enright, A. J. (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36, D154–8.
Droege, M. and Hill, B. (2008) The Genome Sequencer FLX System-longer reads, more applications, straight forward bioinformatics and more complete data sets. J Biotechnol 136, 3–10.
Acknowledgments
ARS and PMP are supported by the Portuguese Foundation for Science and Technology (FCT). We are also thankful to the Centre for Neurosciences of the University of Coimbra for providing a PhD fellowship to ARS and to Biocant for making available the Genome Sequencer FLX. This project was funded by FCT/FEDER project SAU-MMO/55476/2004.
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Soares, A.R., Pereira, P.M., Santos, M.A.S. (2012). Next-Generation Sequencing of miRNAs with Roche 454 GS-FLX Technology: Steps for a Successful Application. In: Fan, JB. (eds) Next-Generation MicroRNA Expression Profiling Technology. Methods in Molecular Biology, vol 822. Humana Press. https://doi.org/10.1007/978-1-61779-427-8_13
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DOI: https://doi.org/10.1007/978-1-61779-427-8_13
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