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Konstantin S. Spirin, Alexander V. Ljubimov, Raquel Castellon, Oryla Wiedoeft, Matthew Marano, Dean Sheppard, M. Cristina Kenney, Donald J. Brown; Analysis of Gene Expression in Human Bullous Keratopathy Corneas Containing Limiting Amounts of RNA. Invest. Ophthalmol. Vis. Sci. 1999;40(13):3108-3115.
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purpose. To validate the use of polymerase chain reaction (PCR)–amplified
full-length cDNA as a substitute for mRNA in nucleic acid array and
gene expression analysis.
methods. Total RNA was isolated from age-matched normal autopsy corneas and
pseudophakic bullous keratopathy (PBK) corneas. Full-length cDNA was
generated and PCR amplified using the Smart cDNA synthesis technology.
Southern blot analysis of this cDNA was compared with Northern blot
analysis of the RNA. Amplified cDNA was used to probe a commercial gene
array. By immunohistochemistry, the expression pattern of several
adhesion molecules represented on the array was assessed.
results. The cDNA produced by the Smart cDNA system gave results very similar to
those of northern blot analysis when examined forβ 2-microglobulin, Rab geranylgeranyl transferase, and
tenascin-C. This cDNA obtained from normal or PBK corneas was labeled
and used to probe a 588 gene array (Clontech). Among other differences,β 6 integrin was detected only with the PBK probe,β
-catenin was markedly elevated in PBK, and β4 integrin
appeared to be reduced in PBK. Immunohistochemical patterns of these
proteins were consistent with the hybridization signals on the gene
conclusions. Smart cDNA synthesis and nucleic acid arrays were combined and
validated for the first time to identify differential gene expression
in normal and diseased corneas. These techniques require very little
RNA such as that equivalent to a half of a single cornea, which is
useful when the amount of tissue is limiting. Altered expression of
adhesive proteins β6 integrin and β-catenin may be
related to the formation of epithelial bullae and microcystic changes
in PBK patients.
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