Summary
As a tool for high-throughput, quantitative gene expression analysis, serial analysis of gene expression (SAGE) is one of the most powerful techniques. However, the short size of tags (14 bp) has hindered the application of SAGE to a vast majority of eukaryotes without sufficient genomic resources, including expressed sequence tag and genome sequences. To overcome this problem, we developed SuperSAGE, which is based on 26-bp tags from complementary DNA (cDNA), using EcoP15I as a tagging enzyme. Because longer cDNA fragments can easily be recovered by 3\(^\prime\)-rapid amplification of cDNA ends (RACE) PCR using primers corresponding to the 26-bp tag sequences in non-model organisms, SuperSAGE allows the identification of novel genes in all eukaryotic organisms, and recommends itself as a useful platform in various fields of biological studies.
Here, we present an updated SuperSAGE protocol, which incorporates several modifications and some recommendations to avoid total failure, particularly in the EcoP15I digestion step.
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Reference
Tan, P. K., Downey, T. J., Spitznagel, E. L., et al. (2003) Evaluation of gene expression measurements from commercial microarray platforms. Nucl. Acids Res. 31, 5676–5684.
Su’arez-Fari nas, M., Noggle, S., Heke, M., Hemmati-Brivanlou, A., and Magnasco, M. O. (2005) Comparing independent microarray studies: the case of human embryonic stem cells. BMC Genomics 6, 99–109.
Ding, C. and Cantor, C. (2003) High-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. Proc. Natl. Acad. Sci. USA 100, 3059–3064.
Brenner, S., Johnson, M., Bridgham, J., et al. (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nature Biotech. 18, 630–634.
Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995) Serial analysis of gene expression. Science 270, 484–487.
Matsumura, H., Reich, S., Ito, A., et al. (2003) Gene expression analysis of plant host-pathogen interactions by SuperSAGE. Proc. Natl. Acad. Sci. USA 100, 15718–15723.
Matsumura, H., Reich, S., Reuter, M., et al. (2004) SuperSAGE: A transcriptome tool for eukaryotic organisms, in SAGE: Current Technologies and Applications (Wang, S. M., ed.). Horizon Scientific Press, Norwich: pp. 77–90.
Matsumura, H., Ito, A., Saitoh, H., et al. (2005) SuperSAGE. Cell. Microbiol. 7, 11–18.
Kim, D-H., Behlke, M. A., Rose S. D., Chang, M-S., Choi, S. and Rossi, J. J. (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotech. 23, 222–226.
Siolas, D., Lerner, C., Burchard, J., et al. (2005) Synthetic shRNAs as potent RNAi triggers. Nat. Biotech. 23, 227–231.
Gowda, M., Jantasuriyarat, C., Dean, R. A., and Wang, G.L. (2004) Robust-LongSAGE (RL-SAGE): a substantially improved LongSAGE method for gene discovery and transcriptome analysis. Plant Physiol. 134, 890–897.
Moncke-Buchner, E., Mackeldanz, P., Kruger, D. H, and Reuter, M. (2004) Overexpression and affinity chromatography purification of the Type III restriction endonuclease EcoP15I for use in transcriptome analysis. J. Biotechnol. 114, 99–106.
Powell, J. (1998) Enhanced concatemer cloning–-a modification to the SAGE (Serial Analysis of Gene Expression) technique. Nucl. Acids Res. 26, 3445–3446.
Acknowledgments
This work was supported by “Program for Promotion of Basic Research Activities for Innovative Biosciences” (Japan), Grant-in-Aid for Young Scientist (A) 1868801 and by “Iwate University 21st, Century COE Program”. Research of P. Winter and G. Kahl is supported by the European Union (contract FOOD CT-2004–506223) and IAEA (CRP 302–041-GFR-8148). Work of D. H. K. and M. R. was supported by Deutsche Forschungsgemeinschaft grant Kr 1293/4.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Matsumura, H., Reuter, M., Krüger, D.H., Winter, P., Kahl, G., Terauchi, R. (2008). SuperSAGE. In: Nielsen, K.L. (eds) Serial Analysis of Gene Expression (SAGE). Methods in Molecular Biology™, vol 387. Humana Press. https://doi.org/10.1007/978-1-59745-454-4_4
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DOI: https://doi.org/10.1007/978-1-59745-454-4_4
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