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Post-mortem iris tissue was pooled from 10 individuals ranging in age from 4-80 years and RNA was extracted. From this pooled sample an aliquot of 60ug of total RNA yielded 2.17ug of mRNA. A directionally cloned cDNA library in the pCMVSPORT6 vector was constructed at Life Technologies, essentially following the protocols of the SuperScript Plasmid System full details of which are contained in the manufacturer's Instruction manual (http://www.lifetech.com/). First strand synthesis was carried out using a Not I primer-adapter [5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCC(T)15-3']. Not I/blunt end inserts were cloned into the Not I/EcoR V sites in the vector. EST analysis was performed on the unamplified library at the NIH Intramural Sequencing Center (NISC).
Nucleotide
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