Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.
To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002531:Cerebellum / Q000502:physiology; D002540:Cerebral Cortex / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004655:Emulsions; D005786:Gene Expression Regulation; D007700:Kinetics; D008251:Macaca / Q000235:genetics*; D008252:Macaca fascicularis / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D019800:Phenol; D010636:Phenols; D013004:Somatostatin / Q000235:genetics
D004655:Emulsions; D010636:Phenols; D019800:Phenol; D013004:Somatostatin; D004247:DNA