Serial analysis of gene expression:SAGE

SAGE

SAGE is a powerful tool that allows the analysis of overall gene expression patterns with digital analysis.

Because SAGE does not require a preexisting clone, it can be used to identify and quantitate new genes as well as known genes.

SAGE PRINCIPLE

A short sequence tag (10-14bp) contains sufficient information to uniquely identify a transcript provided that that the tag is obtained from a unique position within each transcript;

Sequence tags can be linked together to from long serial molecules that can be cloned and sequenced; and

Quantitation of the number of times a particular tag is observed provides the expression level of the corresponding transcript.

SAGE ADVANTAGE

Linking the tags together allows for rapid sequencing analysis of multiple transcripts.

By linking the tags together, only one sequencing event is required to sequence every transcript within the cell.

Makes the task of DNA expression profiling a much easier

SAGE METHODOLOGY

Generate cDNA primed with biotin-oligo(dT)

NlaIII

Linker have restriction site for “tagging enzyme”(BsmFI)

Schematic of SAGE method:

What does the data look like?

TAG COUNT TAG COUNT TAG COUNTCCCATCGTCC 1286 CACTACTCAC 245 TTCACTGTGA 150CCTCCAGCTA 715 ACTAACACCC 229 ACGCAGGGAG 142CTAAGACTTC 559 AGCCCTACAA 222 TGCTCCTACC 140GCCCAGGTCA 519 ACTTTTTCAA 217 CAAACCATCC 140CACCTAATTG 469 GCCGGGTGGG 207 CCCCCTGGAT 136CCTGTAATCC 448 GACATCAAGT 198 ATTGGAGTGC 136TTCATACACC 400 ATCGTGGCGG 193 GCAGGGCCTC 128ACATTGGGTG 377 GACCCAAGAT 190 CCGCTGCACT 127GTGAAACCCC 359 GTGAAACCCT 188 GGAAAACAGA 119CCACTGCACT 359 CTGGCCCTCG 186 TCACCGGTCA 118TGATTTCACT 358 GCTTTATTTG 185 GTGCACTGAG 118ACCCTTGGCC 344 CTAGCCTCAC 172 CCTCAGGATA 114ATTTGAGAAG 320 GCGAAACCCT 167 CTCATAAGGA 113GTGACCACGG 294 AAAACATTCT 161 ATCATGGGGA 110

SAGE APPLICATION

SAGE is useful in comparative expression studies to identify differences in gene expression between two or more cellular sources of RNA.

A big advantage of the method is its speed: all genes expressed at 0.5% of total mRNA in one tissue but not abundant in another could be found in a single day

Approximately 1000 sequence tags from each tissue.

SAGEmap

http://www.sagenet.org/

Sage resources

Sage data