Selective amplification via biotin- and restriction-mediated enrichment (SABRE), a novel selective amplification procedure for detection of differentially expressed mRNAs.
We present a novel subtractive enrichment protocol for the identification of differentially expressed mRNA species. This procedure, called SABRE (selective amplification via biotin- and restriction-mediated enrichment), uses selective streptavidin-biotin affinity and restriction enzyme site reconstitution to enrich for cDNA species more abundant in one population than in another. Analysis of liver cDNA from a mouse strain expressing the neomycin resistance gene demonstrated that this procedure is capable of identifying species present in one population but absent from another. Furthermore, experiments to identify genes with circadian expression patterns in mouse liver demonstrated that SABRE is capable of detecting even modest 2- to 10-fold differences in accumulation of moderately rare mRNA species, representing as little as 0.03% of total mRNA. These experiments identified the gene encoding coumarin 7-hydroxylase as displaying circadian expression in mouse liver.
Lavery
D J
DJ
Department of Molecular Biology, University of Geneva, 30, quai Ernest-Ansermet, CH1211 Geneva 4, Switzerland.
Lopez-Molina
L
L
Fleury-Olela
F
F
Schibler
U
U
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001710:Biotin; D002940:Circadian Rhythm; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D006801:Humans; D008099:Liver / Q000201:enzymology*; D051379:Mice; D006899:Mixed Function Oxygenases / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*
D018076:DNA, Complementary; D012333:RNA, Messenger; D001710:Biotin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6