SuperSAGE

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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 described 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 trancript, 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 (such as e.g. 454 Life Sciences pyrosequencing), hundred thousands or millions of tags can be analyzed in one run, producing very precise gene expression profiles.

The 26 bp tags have a number of advantages over the smaller SAGE 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 genes can be discovered, that escape detection on microarrays.
  • Sense and anti-sense 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; Matsumura et al., 2006).
  • 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.

[edit] References

  1. ^ a b Hideo Matsumura, Stefanie Reich, Akiko Ito, Hiromasa Saitoh, Sophien Kamoun, Peter Winter, Günter Kahl, Monika Reuter, Detlev H. Krüger, and Ryohei Terauchi (2003): "Gene expression analysis of plant host–pathogen interactions by SuperSAGE." PNAS 100:15718–15723. PMID 14676315

[edit] Further reading

  • Matsumura H, Bin Nasir KH, Yoshida K, Ito A, Kahl G, Kruger DH, Terauchi R. (2006): "SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays." Nat Methods 3:469-474. PMID 16721381
  • Coemans B, Matsumura H, Terauchi R, Remy S, Swennen R, Sagi L. (2005): "SuperSAGE combined with PCR walking allows global gene expression profiling of banana (Musa acuminata), a non-model organism." Theor Appl Genet 111:1118-11126. PMID 16133315
  • Nasir KH, Takahashi Y, Ito A, Saitoh H, Matsumura H, Kanzaki H, Shimizu T, Ito M, Fujisawa S, Sharma PC, Ohme-Takagi M, Kamoun S, Terauchi R (2005): "High-throughput in planta expression screening identifies a class II ethylene-responsive element binding factor-like protein that regulates plant cell death and non-host resistance." Plant J. 43:491-505.
  • Matsumura H, Ito A, Saitoh H, Winter P, Kahl G, Reuter M, Kruger DH, Terauchi R. (2005): "SuperSAGE." Cell Microbiol 7:11-18. PMID 15617519

[edit] External links