Serial Analysis of Gene Expression (SAGE), originally described by Velculescu et al. in Science counts polyadenylated transcripts by sequencing a short 14 bp tag at the genes 3’end, adjacent to the last restriction site. A schematic representation of the standard SAGE method is outlined below.
Basic steps in the SAGE protocol
Select cells for profiling. Isolate mRNA using magnetic beads and synthesize cDNA. Cut transcript with anchoring enzyme (NlaIII). Release 14 bp tags from the 3' end, adjacent to the last restriction site with tagging enzyme BsmF1. Pair and ligate tags to form ditags, and concatenate ditags into plasmids for automtic sequencing. Extract and count tags to calculate expression level of each transcript.
For SAGE Genie the ‘anchoring enzyme’ is Nla III. Libraries have been made from both cell lines and tissues and the tag counts (typically >50,000 per RNA sample) are stored for comparison of gene expression. Tag sequence and knowledge of it’s position next to the 3’ most Nla III site is also used to identify the gene from which a particular tag sequence originates.
SAGE Reviews and Protocols
Several reviews (references 2-6) have been published describing the technology. A detailed protocol and other useful information can be obtained through the SAGE Home Page from the Johns Hopkins Oncology Cente. This site also maintains a large set of publications on the use of SAGE technology.
CGAP SAGE Project
To provide quantitative expression levels on a genome wide scale, the Cancer Genome Anatomy Project (CGAP) adapted SAGE in 1999, launching the CGAP SAGE Project (Lal et al.). Over 5 million transcript tags from more than 100 human cell types have been assembled and released to the public domain by this project. This data is also posted in collaboration with the NCBI on the SAGEmap website, where several other tools for the analysis of SAGE data are located.
In 2002, a new and improved and more reliable analysis of tag to gene mapping, called SAGE Genie, was developed to replace the original CGAP SAGE analysis:Boon, K.,Osório, E., Greenhut, S.F., Schaefer, C.F., Shoemaker, J., Polyak, K., Morin, P.J., Buetow, K.H., Strausberg, R.L., de Souza, S.J. and Riggins, G.J. (2002). An anatomy of normal and malignant gene expression. Proc Natl Acad Sci 99 (17):11287-92.In tandem with this altenative analytical approach, new analytical tools have been designed to provide a visual and intutitive representation of gene expression.
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