Analysis of gene expression in human bullous keratopathy corneas containing limiting amounts of RNA.
OBJECTIVE
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 for beta2-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, beta6 integrin was detected only with the PBK probe, beta-catenin was markedly elevated in PBK, and beta4 integrin appeared to be reduced in PBK. Immunohistochemical patterns of these proteins were consistent with the hybridization signals on the gene array.


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 beta6 integrin and beta-catenin may be related to the formation of epithelial bullae and microcystic changes in PBK patients.
Spirin
K S
KS
Department of Surgery, Burns and Allen Research Institute, Cedars-Sinai Medical Center, University of California at Los Angeles Medical School Affiliate, 90048, USA.
Ljubimov
A V
AV
Castellon
R
R
Wiedoeft
O
O
Marano
M
M
Sheppard
D
D
Kenney
M C
MC
Brown
D J
DJ
D015703:Antigens, CD / Q000096:biosynthesis / Q000378:metabolism*; D015139:Blotting, Southern; D003316:Corneal Diseases / Q000235:genetics* / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis; D019084:Fluorescent Antibody Technique, Indirect; D005784:Gene Amplification; D015870:Gene Expression; D006801:Humans; D039641:Integrin beta Chains; D039663:Integrin beta4; D016023:Integrins / Q000096:biosynthesis / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D015534:Trans-Activators; D051176:beta Catenin
D015703:Antigens, CD; C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D039641:Integrin beta Chains; D039663:Integrin beta4; D016023:Integrins; D015345:Oligonucleotide Probes; D015534:Trans-Activators; D051176:beta Catenin; C072170:integrin beta6; D012313:RNA