Abstract
Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10–30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3′-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5′ adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing–compatible cDNA libraries that code for functional ncRNAs within 1 week.
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References
Birney, E. et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007).
Mattick, J.S. & Makunin, I.V. Small regulatory RNAs in mammals. Hum. Mol. Genet. 14 Spec No 1: R121–R132 (2005).
Willingham, A.T. & Gingeras, T.R. TUF love for 'junk' DNA. Cell 125, 1215–1220 (2006).
Huttenhofer, A., Schattner, P. & Polacek, N. Non-coding RNAs: hope or hype? Trends Genet. 21, 289–297 (2005).
Brosius, J. Waste not, want not—transcript excess in multicellular eukaryotes. Trends Genet. 21, 287–288 (2005).
Eddy, S.R. Non-coding RNA genes and the modern RNA world. Nat. Rev. Genet. 2, 919–929 (2001).
Huttenhofer, A. & Schattner, P. The principles of guiding by RNA: chimeric RNA-protein enzymes. Nat. Rev. Genet. 7, 475–482 (2006).
Bartel, D.P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).
Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).
Bachellerie, J.P., Cavaille, J. & Huttenhofer, A. The expanding snoRNA world. Biochimie 84, 775–790 (2002).
Rederstorff, M. et al. RNPomics: defining the ncRNA transcriptome by cDNA library generation from ribonucleo-protein particles. Nucleic Acids Res. 38, e113 (2010).
Huttenhofer, A., Brosius, J. & Bachellerie, J.P. RNomics: identification and function of small, non-messenger RNAs. Curr. Opin. Chem. Biol. 6, 835–843 (2002).
Huttenhofer, A., Cavaille, J. & Bachellerie, J.P. Experimental RNomics: a global approach to identifying small nuclear RNAs and their targets in different model organisms. Methods Mol. Biol. 265, 409–428 (2004).
Huttenhofer, A. & Vogel, J. Experimental approaches to identify non-coding RNAs. Nucleic Acids Res. 34, 635–646 (2006).
Jochl, C. et al. Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis. Nucleic Acids Res. 36, 2677–2689 (2008).
Lung, B. et al. Identification of small non-coding RNAs from mitochondria and chloroplasts. Nucleic Acids Res. 34, 3842–3852 (2006).
Madej, M.J., Alfonzo, J.D. & Huttenhofer, A. Small ncRNA transcriptome analysis from kinetoplast mitochondria of Leishmania tarentolae. Nucleic Acids Res. 35, 1544–1554 (2007).
Mrazek, J., Kreutmayer, S.B., Grasser, F.A., Polacek, N. & Huttenhofer, A. Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells. Nucleic Acids Res. 35, e73 (2007).
Mercer, T.R., Dinger, M.E. & Mattick, J.S. Long non-coding RNAs: insights into functions. Nat. Rev. Genet. 10, 155–159 (2009).
Kapranov, P. et al. Examples of the complex architecture of the human transcriptome revealed by RACE and high-density tiling arrays. Genome Res. 15, 987–997 (2005).
Marioni, J.C., Mason, C.E., Mane, S.M., Stephens, M. & Gilad, Y. RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 18, 1509–1517 (2008).
Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57–63 (2009).
Velculescu, V.E., Zhang, L., Vogelstein, B. & Kinzler, K.W. Serial analysis of gene expression. Science 270, 484–487 (1995).
Fullwood, M.J., Wei, C.L., Liu, E.T. & Ruan, Y. Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses. Genome Res. 19, 521–532 (2009).
Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).
Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).
Huttenhofer, A. et al. RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse. EMBO. J. 20, 2943–2953 (2001).
Acknowledgements
This work was supported by the Medical University of Innsbruck and the Austrian Genome Research (GEN-AU; http://www.gen-au.at/) funding program (grants D-110420-011-013 and D-11420-011-015 to A.H.). We acknowledge M. Lukasser, K. Skreka and M. Erlacher for technical assistance.
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M.R. designed and performed the experiments and wrote the manuscript. A.H. designed the experiments and edited the manuscript.
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Rederstorff, M., Hüttenhofer, A. cDNA library generation from ribonucleoprotein particles. Nat Protoc 6, 166–174 (2011). https://doi.org/10.1038/nprot.2010.186
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DOI: https://doi.org/10.1038/nprot.2010.186
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