[PubMed] [Google Scholar] 29. and RPE degeneration and atrophy and a few drusenoid deposits (9, 10). Materials and Methods Mice.DKO mice and C57BL/6N (WT) mice were bred in-house. These mice were termed DKO in our previous publications (9, 11), and now DKO refers to on an background (7). The study was conducted in compliance with the Association for Research in Vision and Ophthalmology statement for the ethical use of animals. Two of 54 mice died during the experiment from a fight with a mouse in the same cage, so data from these mice were excluded from analysis. All animal experiments were performed under protocols approved by the National Eye Institute (NEI)s Institutional Animal Care and Use Committee. Experimental LRRFIP1 antibody protocol.DKO and WT mice were separated from their mothers at 3 wk of age, randomly assigned to 2 groups, and separately fed the standard diet. Feeding habits were observed and we calculated that the mean consumption of both WT and DKO mice was 4.0 0.2 g/d diet and the mean body weight was 16 0.3 g. The doses of these ingredients in the experimental formula were determined using the human AREDS2 clinical trial dose (12) and converting this to the mouse dose using allometry formulas (A diet). The 2 2 pelleted, purified animal diets used (provided by Dyets) were based on the AIN-93G formulation (13, 14) with several modifications. The isocaloric control diet is (C diet) identical to the AIN-93G diet with the exception of an increased amount of soybean oil (117 vs. 70 g/kg diet) and the experimental diet is identical to the AIN-93G diet with the following modifications: no soybean oil, 1.76 mmol zeaxanthin/kg diet, 17.6 mmol lutein/kg diet, 54.9 mmol EPA/kg diet, and 25.2 mmol DHA/kg diet (Supplemental Table 1). The effective daily dose is: 1.76 were synthesized by SuperArray and supplied as the RT2 Real-Time Gene Expression Assay kit. RT-PCR was performed using previously described methods (14). The levels of the target mRNAs were quantified, using masked procedures, relative to the level of the housekeeping gene, -actin, by the comparative CT method. The formula is the fold of = 10^Ct/standard curve slope, Ct = [Ct (target gene of the tissue) ? Ct (-actin of the tissue)] C [Ct (target gene of the reference) ? Ct AVX 13616 (-actin of the reference)]. In the event that an individual mRNA level was more than 2 SDs above the group when not included, it was considered an outlier and excluded. The results were calculated by using universal total RNA as the reference (SABiosciences). Each sample was analyzed twice. Retina fatty acid analyses.Five eyes in each treatment group were used for retinal fatty acid analyses. Total cellular lipids were extracted from the retina samples as previously described (17). Briefly, retinas were manually homogenized in a small volume of ice-cold buffer (50 mmol/L Trizma, 1 mmol/L EDTA, pH 7.4). After acidification with 0.1 nmol/L HCL, total lipids were extracted with chloroform-methanol (2:1). The organic phase was concentrated under a stream of AVX 13616 nitrogen gas. FAMEs were prepared by base-catalyzed methylation (0.5 mol/L sodium methoxide in methanol) and analyzed using an Agilent Technologies gas chromatograph equipped with 60- 0.25-mm i.d.-fused silica capillary column with a 0.15-value). A2E concentrations (C-DKO vs. A-DKO and C-WT vs. A-WT), gene expression level (A-DKO vs. C-DKO), retinal outer layer thickness (C-WT vs. A-WT and C-DKO vs. A-DKO), serum AVX 13616 PGE2 concentration (A-DKO vs. C-DKO), and retinal fatty acid concentrations (A-WT vs. C-WT and A-DKO vs. C-DKO) were compared after 3 mo of treatment using a 2-tailed unpaired Mann-Whitney U test. Values in the text are presented as median (minimum value, maximum value) (U = Mann-Whitney U value, 0.05 for chi-squared and Mann-Whitney U analyses. XLSTAT software version 2011.4.02 and Microsoft Excel were used for statistical analysis. Results Three independent experiments were performed on DKO and WT mice fed either the control diet (C-DKO and C-WT) or the AREDS2-supplemented diet (A-DKO and A-WT) for.