Subjects were randomly allocated to receive a daily dose of fish oil containing a dose of 1800 mg EPA/DHA, or a high oleic acid sunflower oil (HOSF) for a period of 26 weeks (Lipid Nutrition/ Loders Croklaan, Wormerveer, the Netherlands), in addition to their regular diet. The oils were administered in six soft gelatin capsules daily, each containing 900 mg oil and 2.7 mg tocopherol as antioxidant (Banner Pharmacaps Europe B.V., Tilburg, the Netherlands). The fatty acid composition of the capsules was measured in 20 capsules taken at regular times during the study.
Growth protocol
Healthy men and women of 65 years and over were screened and recruited according to the following exclusion criteria: current or recent (<4 weeks) use of fish oil supplements or intake of fish more than four times per week or more than 800 mg of EPA-DHA from fish per day as estimated by a fish consumption questionnaire, serious liver disease, consumption of more than 4 glasses of alcohol per day, unable to participate as judged by the responsible medical physician, allergy to fish(oil), swallowing problems, or participation in another clinical trial less than 2 months before the start of the trial or at the same time. Additionally, compliance to capsule use during a 2-week placebo run-in period had to be at least 80% on basis of self-report. 302 subjects were included in the study and detailed baseline characteristics of these participants have been described elsewhere (van de Rest, Neurology, 2008) From these participants, 111 subjects were randomly included in the present study. All subjects gave written informed consent to participate in the study and the study protocol was approved by the Medical Ethical Committee of Wageningen University, the Netherlands.
Extracted molecule
total RNA
Extraction protocol
Fasting venous blood samples were collected at baseline and after 26 weeks of intervention. For PBMC isolation four ml of blood was collected,in BD Vacutainer Cell Preparation Tubes with sodium citrate (BD, Breda, The Netherlands). PBMCs were isolated immediately after blood collection according to the manufacturer's instructions. Isolated PBMCs were lysed in Qiagen RLT buffer and homogenized using seringes with 0.4 mm needles, whereafter purified total RNA was isolated using Qiagen RNEasy columns according to the manufacturer's instructions.
Label
biotin
Label protocol
The Ambion MessageAmp aRNA Amplification Kit was used to prepare labelled cRNA from 500ng of total RNA. The protocol was conducted using the reagents provided by Ambion (P/N 1751), as per the manufacturer's instructions.
Hybridization protocol
Hybridisation of 10ug cRNA was done overnight for 16 hours at 45ºC in a Hybridisation Oven 640 (Affymetrix). The protocol is conducted as described in the "Genechip Expression Analysis Technical Manual", section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Target Hybridization) (P/N 701027, revision 5).
Scan protocol
Arrays were scanned on an Affymetrix 3000 7G scanner, as described n the "Genechip Expression Analysis Technical Manual", section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
