An efficient approach for the selective isolation of specific transcripts from complex brain mRNA populations.
As a consequence of the complexity of the nervous system, many characteristic proteins are only expressed at low levels, and their detection and purification often represents a formidable task. A strategy based on liquid hybridizations, which greatly facilitates the identification of the mRNA transcript for this kind of protein, is presented here. A ten-fold-enriched recombinant library was generated from cDNA transcribed from forebrain mRNA after subtraction of cerebellum sequences. Clones specific to forebrain could then be revealed with cerebellum subtracted probes, by colony hybridization. The use of selected cDNA populations greatly enhanced the sensitivity of the screening procedure; clones corresponding to transcripts present at an abundance as low as 0.0005% could still be detected. About 5% of specific clones were recognized with an enriched forebrain probe. Additional clones were revealed with subtracted probes from restricted areas such as cerebral cortex, brainstem, and hippocampus. The important features and potential applications of this approach are discussed.
D000818:Animals; D001923:Brain Chemistry; D002531:Cerebellum / Q000032:analysis; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005625:Frontal Lobe / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D009928:Organ Specificity; D012333:RNA, Messenger / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic
D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D004247:DNA