SuperSAGE.
As a tool for high-throughput, quantitative gene expression analysis, serial analysis of gene expression (SAGE) is one of the most powerful techniques. However, the short size of tags (14 bp) has hindered the application of SAGE to a vast majority of eukaryotes without sufficient genomic resources, including expressed sequence tag and genome sequences. To overcome this problem, we developed SuperSAGE, which is based on 26-bp tags from complementary DNA (cDNA), using EcoP15I as a tagging enzyme. Because longer cDNA fragments can easily be recovered by 3'-rapid amplification of cDNA ends (RACE) PCR using primers corresponding to the 26-bp tag sequences in non-model organisms, SuperSAGE allows the identification of novel genes in all eukaryotic organisms, and recommends itself as a useful platform in various fields of biological studies. Here, we present an updated SuperSAGE protocol, which incorporates several modifications and some recommendations to avoid total failure, particularly in the EcoP15I digestion step.
Matsumura
Hideo
H
Iwate Biotechnology Research Center, Iwate, Japan.
Reuter
Monika
M
Krüger
Detlev H
DH
Winter
Peter
P
Kahl
Günter
G
Terauchi
Ryohei
R
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D005822:Genetic Vectors; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
D018076:DNA, Complementary; C048422:endodeoxyribonuclease NlaIII; D015252:Deoxyribonucleases, Type II Site-Specific