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R. M. Corrales, A. De la Mata, M. Lopez, V. Saez, M. De la Paz, M. Calonge; A New Method for Quantifying Limbal Stem Cell Markers. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6074.
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© ARVO (1962-2015); The Authors (2016-present)
To analyze the expression of 84 genes related to the identification, growth, maintenance, and differentiation of stem cells using a real time reverse transcription (RT2) PCR array (The Human Stem Cell RT² ProfilerTM, SuperArray Bioscience Co.) in healthy adult human corneal and limbal samples.
RNA was isolated from 11 limbal and central cornea epithelial cells. cDNA was synthesized and used for a RT2 PCR array, that combines real time PCR sensitivity and specificity with the ability of microarrays to detect the expression of multiple genes simultaneously. The expression of 84 human genes related to: 1) stem cell specific markers (cell cycle regulators, chromosome and chromatin modulators, genes regulating symmetric/asymmetric cell division, self-renewal markers, cytokines and growth factors, genes regulating cell-cell communication, cell adhesion molecules and metabolic markers); 2) stem cell differentiation markers (embryonic, hematopoietic, mesenchymal and neural cell lineage markers); and 3) signalling pathways important for stem cell maintenance (Notch and Wnt pathways).The comparative Ct method was used for analyzing the results using the cornea as calibrator.
RT2 PCR array indicated an up-regulation in the expression of most limbal genes compared to corneal genes. The relative fold in limbal samples was higher than 7, 4, and 3 for the 9 embryonic, 6 hematopoietic and 6 mesenchymal cell lineage markers, respectively. The highest folds were found for the following genes: CD8A:12.06, DLL3:12.10, ALPI:14.02, ABCG2:15.23, CCND2:15.43, COL1A1:21.83, IGF-1:31.83, K15:87.88 and CXCL12:140.99.
We found new putative limbal stem cell marker genes by RT2 PCR array which require further investigation.
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