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L. Geng, H. Cai, L.V. Del Priore; DIFFERENCES IN GENE EXPRESSION PROFILE OF CULTURED ADULT VERSUS IMMORTALIZED HUMAN RPE . Invest. Ophthalmol. Vis. Sci. 2004;45(13):633.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Immortalized human RPE (cell line ARPE–19) are used widely to draw inferences about the behavior of adult RPE. We have used DNA microarray analysis to compare the gene expression profiles of these two cell types. Methods: Cultured primary RPE from five human donors (age: 48 – 80 years) and ARPE–19 cultured to confluence in five dishes were used for DNA microarray study. Total RNA was isolated using a Qiagen RNeasy Mini Kit. First and second strand cDNA were synthesized with a T7–(dT)24 oligomer for priming and double–stranded cDNA was cleaned with Phase Lock Gels–Phenol/Chloroform extraction and ethanol precipitation. Biotin–labeled antisense cRNA was produced by an in vitro transcription reaction (ENZO BioArray High Yield RNA Transcript Labeling Kit) and incubated with fragmentation buffer (Tris–acetate, KOAc and MgOAcat; 94oC for 35 minutes). Target hybridization, washing, staining and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. Microarray data were treated with normalization, log transformation, statistic determination for "presence" or "absence" for RPE gene expression profile. Clustering analysis, including PCA was used with Affymetrix Microarray Suite 5.0, Genesis 1.30 software. Results: Principle Component Analysis and hierarchic clustering analysis demonstrates that the gene expression profile of the adult RPE and ARPE–19 cluster into two distinct groups with no discernable overlap on the PCA graphs. A scatter plot revealed incomplete overlap in the gene expression profiles of these two cells types. The expression of 6,085 genes (out of 12,600 genes on microarray Human 95UA chip) was detected in ARPE–19 cells, in comparison to expression of only 4,854 genes in adult RPE cells from all 5 human donor eyes. Thus, there were 1,230 genes expressed in ARPE cells but not detected in adult RPE cells whereas 52 genes are expressed only in adult RPE. Of the 500 most abundant expressed genes of adult and immortalized RPE cells, 420 (84%) genes are expressed in both groups. Conclusions:There are some similarities but significant differences in the gene expression profile of cultured adult and immortalized ARPE cells, and it is important to note that some specific genes are only expressed in one of these two groups. These studies suggest caution should be exercised when generalizing results obtained from ARPE–19 to results that would be obtained with adult RPE.
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