SuperSAGE

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SuperSAGE is a further development of the serial analysis of gene expression (SAGE) for the qualitative and quantitative analysis of expressed genes.

As with SAGE be of any transcript (from mRNA , which in cDNA was rewritten), enzymatically a sequence segment excised and as a so-called day ( engl. Won label). If you sequence as many of these tags as possible and count the different tags, you get an answer to the question of which gene was read how often, or how many transcripts of which gene are present in the sample. Due to new high-throughput sequencing methods ( Next Generation Sequencing ), the tag-based analysis of gene expression, also known as Digital Gene Expression Profiling (DGE), is moving more and more to the fore because it does not have the limitations of microarrays and it is possible accurately quantify even extremely rare transcripts.

With SuperSAGE, the type III restriction enzyme EcoP15I generates particularly specific tags that are 26–28  bp long, in contrast to the previous techniques SAGE and LongSAGE with only 14 and 18 bp long tags.

The much longer tags allow a much more precise assignment of the tag to the associated transcript and enable more transcripts to be recognized. The precision of the tags also allows the transcripts of different organisms to be precisely distinguished, so that transcription analyzes of several organisms in the interplay are possible, for example of parasite and host. As in the SAGE protocol, so-called ditags are generated from two tags each, which are amplified using PCR before sequencing .

With modern high-throughput sequencing methods, millions of these tags can now be sequenced very quickly and cheaply, so that a very precise transcription profile is created in which the many rare transcripts, such as transcription factors, can be precisely recorded and counted.

The accuracy of the quantification with a sufficient number of sequenced tags exceeds that of microarrays by far. In addition, with SuperSAGE new transcripts can be identified and samples from eukaryotes with as yet unknown or little known genomes can be examined very precisely.

The long 26–28 bp tags can be used as highly specific primers for further analyzes of new transcripts (e.g. for RACE-PCR ) as probes for identifying clones in a gene bank or even for analyzes with higher throughput directly on a microarray be mocked and thus also use the cost advantage of microarrays.

Individual evidence

  1. ↑ Matsumura, H. et al. (2006): SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays. In: Nat. Methods. 3 (6): 469-474. PMID 16721381 , doi: 10.1038 / nmeth882 .
  2. ↑ Matsumura, H. et al. (2008): SuperSAGE: A Modern Platform for Genome-Wide Quantitative Transcript Profiling. In: Curr Pharm Biotechnol . 9 (5): 368-374. PMID 18855689 , doi: 10.2174 / 138920108785915157 .
  3. ↑ Shendure, J. (2008): The beginning of the end for microarrays. In: Nat. Methods. 5 (7): 585-587. PMID 18587314
  4. ^ Wang, SM (2008): Long-short-long games in mRNA identification: the length matters. In: Curr. Pharm. Biotechnol. 9 (5): 362-367. PMID 18855688 , doi: 10.2174 / 138920108785915166 .