Journal: Acta ophthalmologica

Article Title: Early changes in gene expression induced by blue light irradiation of A2E-laden retinal pigment epithelial cells

doi: 10.1111/aos.12146

Figure Lengend Snippet: Cell death and changes in the gene expression profile induced by blue light exposure of A2E-laden retinal pigment epithelial (RPE) cells were more pronounced with longer exposure time. A2E-laden RPE cells were exposed to blue light for 6, 10, 15 and 20 min. Untreated A2E-free RPE cells, A2E-free RPE cells irradiated for 20 min and A2E-laden RPE cells that had not been exposed to blue light served as controls. The experiment was repeated three times. Cell death was quantified, and RNA was extracted for microarray analysis in each experiment. (A) Per cent of nonviable cells was determined by labelling nuclei in death cells with death red staining and all nuclei with DAPI. Mean ± SEM of three experiments, *p < 0.001, + presence of condition + indicates presence of condition. (B) The gene expression data from the microarrays were analysed by principal component analysis (PCA). Two principal components were extracted, and the projections of the samples were plotted in a two-dimensional score plot using the first and second principal component axes. The first PCA dimension holds most of the variation (21%) in the model. (RPE) refers to A2E-free RPE cells and (RPE + A2E) to A2E-laden RPE cells. The duration of blue light exposure is indicated in minutes (6, 10, 15, 20). (○) RPE, (●) RPE + A2E (□) RPE 20, (◆) RPE + A2E 6, (▲) RPE + A2E 10 (▼) RPE + A2E 15, (■) RPE + A2E 20.

Article Snippet: Cell death was quantified using Dead Red staining, and RNA levels for the entire genome was determined using DNA microarrays (Affymetrix GeneChip Human Genome 2.0 Plus).

Techniques: Gene Expression, Irradiation, Microarray, Staining

Journal: Acta ophthalmologica

Article Title: Early changes in gene expression induced by blue light irradiation of A2E-laden retinal pigment epithelial cells

doi: 10.1111/aos.12146

Figure Lengend Snippet: Quantitative reverse-transcriptase PCR analysis of 12 selected genes confirmed the expression patterns observed in the Affymetrix microarrays. Retinal pigment epithelial (RPE) cells that had accumulated A2E (A2E) were irradiated at 430 nm for 6, 10, 15 and 20 min. RNA was extracted after 6-hr incubation and expression of selected genes was measured by quantitative RT-PCR to validate the microarray data. Controls were A2E-free RPE cells (A2E-free) that were not irradiated or were irradiated for 20 min and cells that had accumulated A2E but were not irradiated (A2E). The RNA level of GAPDH was used as internal amplification control, and Pfaffl method was used to calculate the relative gene expression. (A) Complement Factor I; (B) Complement Factor H; (C) C3; (D) Heat-shock 70 kDa Protein 6; (E) Heat-shock 70 kDa Protein 1A; (F) Heat-shock 70 kDa Protein 1B; (G) Activating Transcription Factor 3; (H) Activating Transcription Factor 4; (I) DNA-damage-inducible transcript 3; (J) CASP8 and FAAD-like apoptosis regulator; (K) Heme Oxygenase 1; (L) CCL2. Mean ± SEM of three experiments. *p < 0.05, **p < 0.01 and ***p < 0.001.

Article Snippet: Cell death was quantified using Dead Red staining, and RNA levels for the entire genome was determined using DNA microarrays (Affymetrix GeneChip Human Genome 2.0 Plus).

Techniques: Reverse Transcription, Expressing, Irradiation, Incubation, Quantitative RT-PCR, Microarray, Amplification, Control, Gene Expression