A novel strategy for identifying differential gene expression: An improved method of differential display analysis.
We propose a novel alternative approach, an advanced method for recently developed strategies, for identifying differentially expressed genes. Firstly, double-stranded cDNAs were digested using Sau3AI and the 3'-end restriction fragments of the cDNA were ligated to a double-stranded adapter. Next, the restriction fragments were directly amplified using several combinations of adapter-specific primers and FITC-labeled oligo dT primers. The selected cDNA fragments were displayed on a polyacrylamide gel. Neither nested PCR nor purification of 3'-end fragments are necessary. We examined the validity of this approach by evaluating gene expression changes during granulocytic differentiation of HL-60 cells. This method can theoretically detect almost all gene expression changes more rapidly and through simpler manipulations than by any other approach.
Kohroki
J
J
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
Tsuchiya
M
M
Fujita
S
S
Nakanishi
T
T
Itoh
N
N
Tanaka
K
K
D002454:Cell Differentiation; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D015870:Gene Expression; D006098:Granulocytes / Q000166:cytology*; D018922:HL-60 Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary