Use of serial analysis of gene expression (SAGE) technology.
Serial analysis of gene expression, or SAGE, is an experimental technique designed to gain a direct and quantitative measure of gene expression. The SAGE method is based on the isolation of unique sequence tags (9-10 bp in length) from individual mRNAs and concatenation of tags serially into long DNA molecules for a lump-sum sequencing. The SAGE method can be applied to the studies exploring virtually any kinds of biological phenomena in which the changes in cellular transcription are responsible. SAGE is a highly competent technology that can not only give a global gene expression profile of a particular type of cell or tissue, but also help us identify a set of specific genes to the cellular conditions by comparing the profiles constructed for a pair of cells that are kept at different conditions. In this review, we present an outline of the original method, several studies achieved by using the method as a major strategic tool, technological difficulties and intrinsic problems that emerged, and improvements and modifications of the method to cope with these drawbacks. We then present our modified SAGE procedure that generates longer sequence tags (14 bp) rather in detail, and the profile (80K profile) derived from HeLa cells that is composed of 80000 tags obtained from a single library. In addition, a series of smaller profiles (2, 4, 10, 20 and 40K) was made by dividing the 80K profile. When we compared these smaller profiles with respect to tag counts for a number of genes, it became apparent that counts of most gene tags increase stably and constantly as the size of profiles increase, while several genes do not. This may be another problem we have to keep in mind, when the profiles are compared for the identification of 'specific genes'.
Yamamoto
M
M
Department of Biochemistry, National Defense Medical College, 3-2 Namiki, Tokorozawa, 359-8513, Saitama, Japan. yamama13@cc.ndmc.ac.jp
Wakatsuki
T
T
Hada
A
A
Ryo
A
A
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015658:HIV Infections / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007158:Immunologic Techniques; D051379:Mice; D017628:Microglia / Q000276:immunology / Q000502:physiology; D009369:Neoplasms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger