出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2015/08/01 12:56:44」(JST)
Serial analysis of gene expression (SAGE) is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu at the Oncology Center of Johns Hopkins University and published in 1995.[1] Several variants have been developed since, most notably a more robust version, LongSAGE,[2] RL-SAGE[3] and the most recent SuperSAGE.[4] Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.
Briefly, SAGE experiments proceed as follows:
The output of SAGE is a list of short sequence tags and the number of times it is observed. Using sequence databases a researcher can usually determine, with some confidence, from which original mRNA (and therefore which gene) the tag was extracted.
Statistical methods can be applied to tag and count lists from different samples in order to determine which genes are more highly expressed. For example, a normal tissue sample can be compared against a corresponding tumour to determine which genes tend to be more (or less) active.
Although SAGE was originally conceived for use in cancer studies, it has been successfully used to describe the transcriptome of other diseases and in a wide variety of organisms.
In 1979 teams at Harvard and Caltech extended the basic idea of making DNA copies of mRNAs in vitro to amplifying a library of such in bacterial plasmids[5]
In 1982-3, the idea of selecting random or semi-random clones from such a cDNA library for sequencing was explored by Greg Sutcliffe and coworkers.[6] and Putney et al. who sequenced 178 clones from a rabbit muscle cDNA library[7]
In 1991 Adams and co-workers coined the term Expressed Sequence Tag (EST) and initiated more systematic sequencing of cDNAs as a project (starting with 600 brain cDNAs).[8] The identification of ESTs has proceeded rapidly, with approximately 72.6 million ESTs now available in public databases (e.g. GenBank 11 May 2011, all species).
In 1995, the idea of reducing the tag length from 100 to 800 bp down to tag length of 10 to 22 bp helped reduce the cost of mRNA surveys.[1]
The general goal of the technique is similar to the DNA microarray. However, SAGE sampling is based on sequencing mRNA output, not on hybridization of mRNA output to probes, so transcription levels are measured more quantitatively than by microarray. In addition, the mRNA sequences do not need to be known a priori, so genes or gene variants which are not known can be discovered. Microarray experiments are much cheaper to perform, so large-scale studies do not typically use SAGE. Quantifying gene expressions is more exact in SAGE because it involves directly counting the number of transcripts whereas spot intensities in microarrays fall in non-discrete gradients and are prone to background noise.
MicroRNAs, or miRNAs for short, are small (~22nt) segments of RNA which have been found to play a crucial role in gene regulation. One of the most commonly used methods for cloning and identifying miRNAs within a cell or tissue was developed in the Bartel Lab and published in a paper by Lau et al. (2001). Since then, several variant protocols have arisen, but most have the same basic format. The procedure is quite similar to SAGE: The small RNA are isolated, then linkers are added to each, and the RNA is converted to cDNA by RT-PCR. Following this, the linkers, containing internal restriction sites, are digested with the appropriate restriction enzyme and the sticky ends are ligated together into concatamers. Following concatenation, the fragments are ligated into plasmids and are used to transform bacteria to generate many copies of the plasmid containing the inserts. Those may then be sequenced to identify the miRNA present, as well as analysing expression levels of a given miRNA by counting the number of times it is present, similar to SAGE.
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| リンク元 | 「SAGE」 |
| 関連記事 | 「serial」「expression」「analysis」 |