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Preparation of Full-Length cDNA Libraries: Focus on Metazoans

  • Masako Harada
  • Yoshihide Hayashizaki
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 533)

Abstract

Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.

Key words

mRNA metazoan full-length cDNA CTAB-urea method cap-trapper method biotinylation of diol groups of mRNA normalization and subtraction of cDNA 

Notes

Acknowledgments

This work was supported by a Research Grant for the RIKEN Genome Exploration Research Project to Y.H and a grant of the Genome Network Project, both from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.

We would like to thank Dr. Carninci P. for his advice, and also the colleagues in the Genome Exploration Research Group at the Genomic Sciences Center (GSC), RIKEN Yokohama Institute, for all the support.

References

  1. 1.
    Carninci, P., Nishiyama, Y., Westover, A., Itoh, M., Nagaoka, S., Sasaki, N., Okazaki, Y., Muramatsu, M. & Hayashizaki, Y. (1998). Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. Proc Natl Acad Sci USA 95, 520–4.PubMedCrossRefGoogle Scholar
  2. 2.
    Carninci, P., Kvam, C., Kitamura, A., Ohsumi, T., Okazaki, Y., Itoh, M., Kamiya, M., Shibata, K., Sasaki, N., Izawa, M., Muramatsu, M., Hayashizaki, Y. & Schneider, C. (1996). High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 37, 327–36.PubMedCrossRefGoogle Scholar
  3. 3.
    Carninci, P., Shibata, Y., Hayatsu, N., Itoh, M., Shiraki, T., Hirozane, T., Watahiki, A., Shibata, K., Konno, H., Muramatsu, M. & Hayashizaki, Y. (2001). Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis. Genomics 77, 79–90.PubMedCrossRefGoogle Scholar
  4. 4.
    Carninci, P., Shibata, Y., Hayatsu, N., Sugahara, Y., Shibata, K., Itoh, M., Konno, H., Okazaki, Y., Muramatsu, M. & Hayashizaki, Y. (2000). Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res 10, 1617–30.PubMedCrossRefGoogle Scholar
  5. 5.
    Sugahara, Y., Carninci, P., Itoh, M., Shibata, K., Konno, H., Endo, T., Muramatsu, M. & Hayashizaki, Y. (2001). Comparative evaluation of 5'-end-sequence quality of clones in CAP trapper and other full-length-cDNA libraries. Gene 263, 93–102.PubMedCrossRefGoogle Scholar
  6. 6.
    Cheng, S., Fockler, C., Barnes, W. M. & Higuchi, R. (1994). Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci USA 91, 5695–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Del Sal, G., Manfioletti, G. & Schneider, C. (1989). The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. Biotechniques 7, 514–20.PubMedGoogle Scholar
  8. 8.
    Van Gilst, M. R., Hadjivassiliou, H., Jolly, A. & Yamamoto, K. R. (2005). Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans. PLoS Biol 3, e53.PubMedCrossRefGoogle Scholar
  9. 9.
    Jing, T., Wang, Z., Liu, K. & Qi, F. (2006). A modified one-step procedure for rapid RNA isolation from insect. J Forestry Res 17, 129–31.CrossRefGoogle Scholar
  10. 10.
    Dreyfuss, G., Matunis, M. J., Pinol-Roma, S. & Burd, C. G. (1993). hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem 62, 289–321.PubMedCrossRefGoogle Scholar
  11. 11.
    Kawai, J., Shinagawa, A., Shibata, K., Yoshino, M., Itoh, M., Ishii, Y., Arakawa, T., Hara, A., Fukunishi, Y., Konno, H., Adachi, J., Fukuda, S., Aizawa, K., Izawa, M., Nishi, K., Kiyosawa, H., Kondo, S., Yamanaka, I., Saito, T., Okazaki, Y., Gojobori, T., Bono, H., Kasukawa, T., Saito, R., Kadota, K., Matsuda, H., Ashburner, M., Batalov, S., Casavant, T., Fleischmann, W., Gaasterland, T., Gissi, C., King, B., Kochiwa, H., Kuehl, P., Lewis, S., Matsuo, Y., Nikaido, I., Pesole, G., Quackenbush, J., Schriml, L. M., Staubli, F., Suzuki, R., Tomita, M., Wagner, L., Washio, T., Sakai, K., Okido, T., Furuno, M., Aono, H., Baldarelli, R., Barsh, G., Blake, J., Boffelli, D., Bojunga, N., Carninci, P., de Bonaldo, M. F., Brownstein, M. J., Bult, C., Fletcher, C., Fujita, M., Gariboldi, M., Gustincich, S., Hill, D., Hofmann, M., Hume, D. A., Kamiya, M., Lee, N. H., Lyons, P., Marchionni, L., Mashima, J., Mazzarelli, J., Mombaerts, P., Nordone, P., Ring, B., Ringwald, M., Rodriguez, I., Sakamoto, N., Sasaki, H., Sato, K., Schonbach, C., Seya, T., Shibata, Y., Storch, K. F., Suzuki, H., Toyo-oka, K., Wang, K. H., Weitz, C., Whittaker, C., Wilming, L., Wynshaw-Boris, A., Yoshida, K., Hasegawa, Y., Kawaji, H., Kohtsuki, S. & Hayashizaki, Y. (2001). Functional annotation of a full-length mouse cDNA collection. Nature 409, 685–90.PubMedCrossRefGoogle Scholar
  12. 12.
    Piche, C. & Schernthaner, J. P. (2005). Optimization of in vitro transcription and full-length cDNA synthesis using the T4 bacteriophage gene 32 protein. J Biomol Tech 16, 239–47.PubMedGoogle Scholar
  13. 13.
    Carninci, P., Shiraki, T., Mizuno, Y., Muramatsu, M. & Hayashizaki, Y. (2002). Extra-long first-strand cDNA synthesis. Biotechniques 32, 984–5.PubMedGoogle Scholar
  14. 14.
    Shibata, Y., Carninci, P., Sato, K., Hayatsu, N., Shiraki, T., Ishii, Y., Arakawa, T., Hara, A., Ohsato, N., Izawa, M., Aizawa, K., Itoh, M., Shibata, K., Shinagawa, A., Kawai, J., Ota, Y., Kikuchi, S., Kishimoto, N., Muramatsu, M. & Hayashizaki, Y. (2001). Removal of polyA tails from full-length cDNA libraries for high-efficiency sequencing. Biotechniques 31, 1042, 1044, 1048–9.Google Scholar
  15. 15.
    Nelson, P. S., Papas, T. S. & Schweinfest, C. W. (1993). Restriction endonuclease cleavage of 5-methyl-deoxycytosine hemimethylated DNA at high enzyme-to-substrate ratios. Nucleic Acids Res 21, 681–6.PubMedCrossRefGoogle Scholar
  16. 16.
    Carninci, P., Westover, A., Nishiyama, Y., Ohsumi, T., Itoh, M., Nagaoka, S., Sasaki, N., Okazaki, Y., Muramatsu, M., Schneider, C. & Hayashizaki, Y. (1997). High efficiency selection of full-length cDNA by improved biotinylated cap trapper. DNA Res 4, 61–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Carninci, P. (2002). Preparation of Representative Full-length cDNA Libraries. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.Google Scholar
  18. 18.
    Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd edition. 3 vols, Laboratory Press, Cold Spring Harbor.Google Scholar
  19. 19.
    Carninci, P., Waki, K., Shiraki, T., Konno, H., Shibata, K., Itoh, M., Aizawa, K., Arakawa, T., Ishii, Y., Sasaki, D., Bono, H., Kondo, S., Sugahara, Y., Saito, R., Osato, N., Fukuda, S., Sato, K., Watahiki, A., Hirozane-Kishikawa, T., Nakamura, M., Shibata, Y., Yasunishi, A., Kikuchi, N., Yoshiki, A., Kusakabe, M., Gustincich, S., Beisel, K., Pavan, W., Aidinis, V., Nakagawara, A., Held, W. A., Iwata, H., Kono, T., Nakauchi, H., Lyons, P., Wells, C., Hume, D. A., Fagiolini, M., Hensch, T. K., Brinkmeier, M., Camper, S., Hirota, J., Mombaerts, P., Muramatsu, M., Okazaki, Y., Kawai, J. & Hayashizaki, Y. (2003). Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia. Genome Res 13, 1273–89.PubMedCrossRefGoogle Scholar
  20. 20.
    Anderson, M. & Young, B. D. (1985). Quantitative Analysis of Solution Hybridisation. Nucleic Acids Hybridisation, a practical approach, IRL Press, Oxford.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Masako Harada
    • 1
  • Yoshihide Hayashizaki
    • 1
    • 2
  1. 1.Genome Exploration Research GroupGenomic Sciences Center (GSC), RIKENYokohamaJapan
  2. 2.Genome Science Laboratory, RIKENWakoJapan

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