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G Maraini, R Ruotolo, C Rivetti, R Percudani, S Ottonello; DNA Array Analysis of Gene Expression in the Lens Epithelium of Pure Nuclear Cataract and Transparent Human Lenses . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3594.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To compare gene expression in lens epithelia from age-related nuclear cataracts and transparent human lenses. Methods:Capsulorrhexis epithelium-capsule samples were obtained at surgery from eyes undergoing cataract extraction for age-related nuclear cataract. Lens opacities were graded in mydriasis at the slit-lamp before surgery according to LOCS II. All cataractous lenses included in this study had a nuclear opacity grade ≷2 and coexisting cortical or posterior subcapsular grade < 1. Epithelium-capsule samples from transparent lenses were obtained with the same technique used for surgical capsulorrhexis, during multi-organ explant procedures. Samples were immediately frozen in liquid nitrogen and stored at - 90° C. Pools of 10-15 capsulorrhexis samples were utilized for RNA extraction. After extraction with RNazol B, total RNA (2-5 micrograms) was converted to single stranded cDNA and 33P-labeled by reverse transcription with the MMLV enzyme. The radiolabeled cDNA was then hybridized to a human DNA macroarray containing a total of 4132 "named" human genes (GF211; Research Genetics/Invitrogen) following the manufacturer's instructions, visualized by phosphorimaging and quantitatively analyzed with the Pathway3 program. Results:Approximately 20% of the cDNAs spotted on the "non-lens specific" array employed for this study gave above background hybridization signals. There was a good overall agreement between the 10 mRNAs most expressed in normal and cataractous lens epithelia, the three top messengers being crystallin alpha B, a yeast SSM4-related messenger (coding for a protein supposedly involved in RNA turnover), and vimentin, About 10 % of the overall set of hybridization-positive mRNAs was differentially modulated in cataractous vs. transparent lens epithelia. Interestingly, the two mRNAs most strongly downregulated in cataractous epithelia (more than 10-fold) code for crystallin beta A1 and beta A4. Conclusion:These preliminary data suggest that potentially meaningful information as to the mRNA expression reprogramming that accompanies cataract development can be obtained from the analysis of non-specialized gene arrays. While an obvious limitation of this approach is the lack of strictly lens specific target genes, the utilization of a non-specialized gene set may shed light on the role of known or as yet uncharacterized (non-lens specific) gene products in human, age-related cataract formation.
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