| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 From the Ophthalmology Research Laboratories, Department of Surgery, Burns and Allen Research Institute, Cedars-Sinai Medical Center, University of California at Los Angeles Medical School Affiliate, Los Angeles, California; 2 Molecular Dynamics, Sunnyvale, California; and 3 Lung Biology Center, University of California at San Francisco.
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 for ß2-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, ß6 integrin was detected only with the PBK probe, ß-catenin was markedly elevated in PBK, and ß4 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 ß6 integrin and ß-catenin may be related to the formation of epithelial bullae and microcystic changes in PBK patients.
This article has been cited by other articles:
|
|
 |
|
  M. Saghizadeh, A. A. Kramerov, J. Tajbakhsh, A. M. Aoki, C. Wang, N.-N. Chai, J. Y. Ljubimova, T. Sasaki, G. Sosne, M. R. J. Carlson, et al. Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas Invest. Ophthalmol. Vis. Sci., October 1, 2005; 46(10): 3604 - 3615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Kenney, M. Chwa, S. R. Atilano, A. Tran, M. Carballo, M. Saghizadeh, V. Vasiliou, W. Adachi, and D. J. Brown Increased Levels of Catalase and Cathepsin V/L2 but Decreased TIMP-1 in Keratoconus Corneas: Evidence that Oxidative Stress Plays a Role in This Disorder Invest. Ophthalmol. Vis. Sci., March 1, 2005; 46(3): 823 - 832. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Brown, B. Lin, and B. Holguin Expression of Neuregulin 1, a Member of the Epidermal Growth Factor Family, Is Expressed as Multiple Splice Variants in the Adult Human Cornea Invest. Ophthalmol. Vis. Sci., September 1, 2004; 45(9): 3021 - 3029. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ivashuta, K. Uchiyama, M. Gau, and Y. Shimamoto Linear amplification coupled with controlled extension as a means of probe amplification in a cDNA array and gene expression analysis during cold acclimation in alfalfa (Medicago sativa L.) J. Exp. Bot., February 1, 2002; 53(367): 351 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Fink, S. Kohlhoff, M. M. Stein, J. Hanze, N. Weissmann, F. Rose, E. Akkayagil, D. Manz, F. Grimminger, W. Seeger, et al. cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue Am. J. Pathol., January 1, 2002; 160(1): 81 - 90. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. V. Ljubimov, M. Saghizadeh, R. Pytela, D. Sheppard, and M. C. Kenney Increased Expression of Tenascin-C-binding Epithelial Integrins in Human Bullous Keratopathy Corneas J. Histochem. Cytochem., November 1, 2001; 49(11): 1341 - 1350. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhang, B. D. Price, S. Tetradis, S. Chakrabarti, G. Maulik, and G. M. Makrigiorgos Reproducible and inexpensive probe preparation for oligonucleotide arrays Nucleic Acids Res., July 1, 2001; 29(13): e66 - e66. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C S SETHI, T A BAILEY, P J LUTHERT, and N H V CHONG Matrix metalloproteinase biology applied to vitreoretinal disorders Br. J. Ophthalmol., June 1, 2000; 84(6): 654 - 666. [Full Text]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
