Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002. Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.1% fungal oil (providing 0.5 dietary wt% AA and 1.0en%). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily. Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France). Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h. After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hepatic gene expression analysis. From these 5 mice, livers were dissected according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), which consisted of carefully dissecting out the liver, rinsing with 70% EtOH to remove adhering blood, trimming of adhering connective tissue with scissors, cutting into 100-150 mg subsections, freezing in liquid N2, and storing at -80°C until gene expression and lipid analysis. Hepatic lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5). Liver tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hepatic RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.
