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P Gonzalez, P Challa, M Caballero, DL Epstein; Generation of Gene Arrays from Human Trabecular Meshwork cDNA Libraries . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3959.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To generate a human trabecular meshwork (HTM) cDNA array for the identification of genes preferentially expressed in the different cell types of the outflow pathway. Methods:The anterior segments of three eye donors were fixed in RNAlater solution less than 5 hours postmortem. The trabecular meshwork was dissected and an independent cDNA library was generated from each donor using the pCR-XL-TOPO vector. White colonies were robotically picked and plated in 386 well plates. Arrays were generated by spotting 27,648 independent clones in nylon membranes. To analyze the quality of the cDNA libraries, 30 clones were randomly selected to measure the insert size and to sequence both ends. Results:The average insert size of the arrayed clones was 1300 bp. Of the clones representing known genes, 47% included the full length cDNA sequence. None of the genes identified was represented by more than one clone. Less than 14% of the clones corresponded to mitochondrial DNA transcripts or non informative inserts. Conclusion:Three cDNA libraries from HTM have been arrayed in nylon membranes. These arrays should provide a useful complement to commercial arrays by providing a more complete representation of those genes with a pattern of expression restricted to the outflow pathway.
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