Abstract
Digital gene expression (DGE) profiling techniques are playing an eminent role in the detection, localization, and differential expression quantification of many small RNA species, including microRNAs (1–3). Procedures in small RNA library preparation techniques typically include adapter ligation by RNA ligase, followed by reverse transcription and amplification by PCR. This chapter describes three protocols that were successfully applied to generate small RNA sequencing SOLiDTM libraries. The Ambion SREKTM-adopted protocol can be readily used for multiplexing samples; the modban-based protocol is cost-efficient, but biased toward certain microRNAs; the poly(A)-based protocol is less biased, but less precise because of the A-tail that is introduced. In summary, each of these protocols has its advantages and disadvantages with respect to the ease of including barcodes, costs, and outcome.
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
Berezikov, E., Cuppen, E., and Plasterk, R. H. (2006) Approaches to microRNA discovery Nat Genet 38 Suppl, S2–7.
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, 1193–207.
Tarasov, V., Jung, P., Verdoodt, B., Lodygin, D., Epanchintsev, A., Menssen, A., et al. (2007) Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest Cell Cycle 6, 1586–93.
Shendure, J. and Ji, H. (2008) Next-generation DNA sequencing Nat Biotechnol 26, 1135–45.
Kawahara, Y., Zinshteyn, B., Sethupathy, P., Iizasa, H., Hatzigeorgiou, A. G., and Nishikura, K. (2007) Redirection of silencing targets by adenosine-to-inosine editing of miRNAs Science 315, 1137–40.
Linsen, S. E., de Wit, E., de Bruijn, E., and Cuppen, E. (2010) Small RNA expression and strain specificity in the rat BMC Genomics 11, 249.
Linsen, S. E., de Wit, E., Janssens, G., Heater, S., Chapman, L., Parkin, R. K., et al. (2009) Limitations and possibilities of small RNA digital gene expression profiling Nat Methods 6, 474–6.
Chen, Y., Gelfond, J. A., McManus, L. M., and Shireman, P. K. (2009) Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis BMC Genomics 10, 407.
Willenbrock, H., Salomon, J., Søkilde, R., Barken, K. B., Hansen, T. N., Nielsen, F. C., et al. (2009) Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing RNA 15, 2028–34.
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.
Kurata, S., Ohtsuki, T., Suzuki, T., and Watanabe, K. (2003) Quick two-step RNA ligation employing periodate oxidation Nucleic Acids Res 31, e145.
de Hoon, M. J., Taft, R. J., Hashimoto, T. Kanamori-Katayama, M., Kawaji, H., Kawano, M., et al. (2010) Cross-mapping and the identification of editing sites in mature microRNAs in high-throughput sequencing libraries Genome Res 20, 257–64.
Acknowledgements
The authors would like to thank Ewart de Bruijn and Nico Lansu for critically reading and contributing to this manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Linsen, S.E.V., Cuppen, E. (2012). Methods for Small RNA Preparation for Digital Gene Expression Profiling by Next-Generation Sequencing. 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_14
Download citation
DOI: https://doi.org/10.1007/978-1-61779-427-8_14
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-426-1
Online ISBN: 978-1-61779-427-8
eBook Packages: Springer Protocols