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Serial Analysis of Gene Expression 271
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Serial Analysis of Gene Expression
Karin A. Oien
1. Introduction
Serial analysis of gene expression (SAGE ) is a patented, large-scale mRNAprofiling
technology that produces comprehensive, quantitative, and reproducible
gene expression profiles (originally described in refs. 1 and 2). Unlike the alternative
technologies of differential display and subtractive hybridization, SAGE produces a
full catalog of all transcripts, not only differentially expressed genes, and unlike smaller
arrays, SAGE needs no assumptions about the genes that are likely to be expressed,
thus allowing the identification of novel genes (are among many excellent reviews, see
refs. 3–5). SAGE is based on generating clones of concatenated (linked) short sequence
tags derived from mRNA from the target cells or tissue (Fig. 1).
Each tag is 9- or 10-bp long and represents one mRNA and each clone insert contains
up to 40 tags joined serially. Therefore, sequencing of multiple concatenates describes
the pattern and abundance of mRNA, with an improvement in efficiency of up to
40-fold compared with conventional analysis of expressed sequence tags. The mRNA
transcript corresponding to the short SAGE tag is identified from genetic databases
using appropriate software.
The SAGE method was developed in 1995 by Velculescu et al. (1) of the Kinzler and
Vogelstein laboratory at Johns Hopkins University, from where the SAGE protocol,
software, and updates are available to academic investigators for noncommercial use
via http://www.sagenet.org/sage_protocol.htm. For commercial purposes, the user
should contact Genzyme Corporation, who own and license the SAGE technology.
This and other useful web sites are listed in Table 1. Since the first report of SAGE,
many technical modifications have been described. Some enable the use of smaller
amounts of starting material (3,6–10), whereas others have improved the efficiency
of intermediary SAGE reactions (11–14). Invitrogen has also released a kit called
I-SAGE , which provides all of the numerous reagents required in a high-quality
form.
The Johns Hopkins protocol is 27 pages long, and Invitrogen’s online I-SAGE
instructions contain 73 pages. This is because SAGE involves many stages, each using
well-established and relatively straightforward molecular biological techniques, but any
of which can go wrong! This article thus provides concise instructions with an emphasis
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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