Lavery D J, Lopez-Molina L, Fleury-Olela F, Schibler U
Department of Molecular Biology, University of Geneva, 30, quai Ernest-Ansermet, CH1211 Geneva 4, Switzerland.
Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6831-6. doi: 10.1073/pnas.94.13.6831.
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.
我们提出了一种用于鉴定差异表达mRNA种类的新型消减富集方案。该程序称为SABRE(通过生物素和限制介导的富集进行选择性扩增),利用选择性链霉亲和素-生物素亲和力和限制酶位点重建来富集在一个群体中比另一个群体中更丰富的cDNA种类。对表达新霉素抗性基因的小鼠品系的肝脏cDNA分析表明,该程序能够鉴定出存在于一个群体中但在另一个群体中不存在的种类。此外,在小鼠肝脏中鉴定具有昼夜表达模式基因的实验表明,SABRE能够检测到中度稀有mRNA种类积累中低至2至10倍的差异,这些mRNA种类仅占总mRNA的0.03%。这些实验确定编码香豆素7-羟化酶的基因在小鼠肝脏中呈现昼夜表达。