SuperSAGE
SuperSAGE is the most advanced derivate of the serial analysis of gene expression technology (SAGE) for the analysis of expressed genes in eukaryotic organisms (gene expression profiling). Like in SAGE, a specific tag from each transcribed gene is recovered. By sequencing and counting as many tags as possible, the transcription profile, stating what gene is transcribed and how often, becomes apparent. SuperSAGE uses the type III-endonuclease EcoP15I of phage P1, to cut 26 bp long sequence tags from each transcript's cDNA, expanding the tag-size by at least 6 bp as compared to the predecessor techniques SAGE and LongSAGE.[1] The longer tag-size allows for a more precise allocation of the tag to the corresponding transcript, because each additional base increases the precision of the annotation considerably.
Like in the original SAGE protocol, so-called ditags are formed, using blunt-ended tags. However, SuperSAGE avoids the bias observed during the less random LongSAGE 20 bp ditag-ligation.
By direct sequencing with modern high-throughput sequencing techniques (next-generation sequencing, i.e. pyrosequencing), hundred thousands or millions of tags can be analyzed simultaneously, producing very precise and quantitative gene expression profiles. Therefore, tag-based gene expression profiling also called "digital gene expression profiling" (DGE) can today provide most accurate transcription profiles that overcome the limitations of microarrays.[2]
The 26 bp tags have a number of advantages over the smaller tags:
- Most notably due to the exact annotation of SuperSAGE tags (at least 10,000 times more accurate than LongSAGE tags), a substantially increased number of transcripts can be differentiated.
- Many different transcript isoforms (representing alternatively spliced transcripts) can be found.
- Novel transcripts (i.e. non-coding RNA) can be discovered, that escape detection on microarrays.
- Sense and antisense transcripts and their different regulation can be detected.
- Due to the exact annotation of the 26 bp tags, two or more interacting organisms (parasite-host, pathogen-host) can be analyzed simultaneously.[1]
- The 26 bp tags can directly be spotted onto microarrays, and candidate transcripts be combined to produce focused microarrays (i.e. microarrays loaded only with genes, that are relevant for a specific process).[3]
- The 26 bp tag allows the design of highly specific primers for downstream PCR (like for 3’- or 5’-RACE) or of specific probes for the identification of clones from a cDNA library.
Very precise and comprehensive gene expression profiles of any eukaryotic organism can therefore be established, which in many regards are superior to microarrays. Each and every transcript can be quantified by counting the tags in a SuperSAGE library such that quantitative genetics is readily possible with SuperSAGE.
References
- 1 2 Matsumura, H.; Reich, S.; Ito, A.; Saitoh, H.; Kamoun, S.; Winter, P.; Kahl, G.; Reuter, M.; Krüger, D.; Terauchi, R. (2003). "Gene expression analysis of plant host-pathogen interactions by SuperSAGE". Proceedings of the National Academy of Sciences 100 (26): 15718–15723. Bibcode:2003PNAS..10015718M. doi:10.1073/pnas.2536670100. PMC 307634. PMID 14676315.
- ↑ Shendure, J. (2008). "The beginning of the end for microarrays?". Nature Methods 5 (7): 585–7. doi:10.1038/nmeth0708-585. PMID 18587314.
- ↑ Matsumura, H.; Bin Nasir, K. H.; Yoshida, K.; Ito, A.; Kahl, G. �N.; Krüger, D. H.; Terauchi, R. (2006). "SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays". Nature Methods 3 (6): 469–74. doi:10.1038/nmeth882. PMID 16721381. replacement character in
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Further reading
- Sharbel, T. F.; Voigt, M. L.; Corral, J. M.; Galla, G.; Kumlehn, J.; Klukas, C.; Schreiber, F.; Vogel, H.; Rotter, B. (2010). "Apomictic and Sexual Ovules of Boechera Display Heterochronic Global Gene Expression Patterns". The Plant Cell Online 22 (3): 655. doi:10.1105/tpc.109.072223.
- Gilardoni, P. A.; Schuck, S.; Jüngling, R.; Rotter, B.; Baldwin, I. T.; Bonaventure, G. (2010). "SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses". BMC Plant Biology 10: 66. doi:10.1186/1471-2229-10-66. PMC 3095340. PMID 20398280.
- Raftery, M. J.; Möncke-Buchner, E.; Matsumura, H.; Giese, T.; Winkelmann, A.; Reuter, M.; Terauchi, R.; Schönrich, G.; Krüger, D. H. (2009). "Unravelling the interaction of human cytomegalovirus with dendritic cells by using SuperSAGE". Journal of General Virology 90 (Pt 9): 2221–2233. doi:10.1099/vir.0.010538-0. PMID 19439557.
- Gowda, M.; Jantasuriyarat, C.; Dean, R.; Wang, G. (2004). "Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis". Plant Physiology 134 (3): 890–7. doi:10.1104/pp.103.034496. PMC 389912. PMID 15020752.