Abstract
A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using “linear models for microarray data” (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.
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References
Ying, S.-Y. (2003) Complementary DNA libraries: an overview, in Generation of cDNA Libraries: Methods and Protocols in Molecular Biology, vol. 221, (Ying, S.-Y. ed.), Humana Press, Totowa, NJ, pp. 1–12.
Diatchenko, L., Lukyanov, S., Lau, Y. F., and Siebert, P. D. (1999) Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes. Methods Enzymol. 303, 349–380.
Bonaldo, M. F., Lennon, G., and Soares, M. B. (1996) Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res. 6, 791–806.
Zhulidov, P. A., Bogdanova, E. A., Shcheglov, A. S., et al. (2004) Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Res. 32, E37.
Mahalingam, R., Gomez-Buitrago, A., Eckardt, N., et al. (2003) Characterizing the stress/defense transcriptome of Arabidopsis. Genome Biol. 4, R20.1–R20.14.
Smyth, G. K. (2004) Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statist. Appl. Genet. Mol. Biol. 3, 1–26.
Yang, G. P., Ross, D. T., Kuang, W. W., Brown, P. O., and Weigel, R. J. (1999) Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Res. 27, 1517–1523.
Vallee, M., Gravel, C., Palin, M. F., et al. (2005) Identification of novel and known oocyte-specific genes using complementary DNA subtraction and microarray analysis in three different species. Biol. Reprod. 73, 63–71.
Hein, I., Campbell, E. I., Woodhead, M., et al. (2004) Characterisation of early transcriptional changes involving multiple signalling pathways in the a13 barley interaction with powdery mildew (Blumeria graminis f. sp. hordei). Planta 218, 803–813.
Cao, W., Epstein, C., Liu, H., et al. (2004) Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study. BMC Genomics 5, 26.
Diatchenko, L., Lau, Y. F., Campbell, A. P., et al. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025–6030.
Van den Berg, N., Crampton, B. G., Hein, I., Birch, P. R., and Berger, D. K. (2004) High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis. Biotechniques 37, 818–824.
Dudoit, S., Yang, Y. H., Callow, M. J., and Speed, T. P. (2002) Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Statistica Sinica 12, 111–139.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Yang, Y. H. and Thorne, N. P. (2003) In Goldstein, D. R. (ed.), Science and Statistics: A Festschrift for Terry Speed. pp. 403–418.
Wit, E. and McClure, J. (eds.) (2004) Statistics for Microarrays: Design, Analysis and Inference. Wiley, Chichester, UK.
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© 2007 Humana Press Inc., Totowa, NJ
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Berger, D.K., Crampton, B.G., Hein, I., Vos, W. (2007). Screening of cDNA Libraries on Glass Slide Microarrays. In: Rampal, J.B. (eds) Microarrays. Methods in Molecular Biology, vol 382. Humana Press. https://doi.org/10.1007/978-1-59745-304-2_12
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DOI: https://doi.org/10.1007/978-1-59745-304-2_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-944-4
Online ISBN: 978-1-59745-304-2
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