The study design consisted of two phases. Phase 1 (days 1-14): controlled dietary intervention in which each of participants had to remain weight stable. Each of the participants was provided with a diet containing approximately 90% of their estimated total daily energy requirement. The remaining 10% was chosen from a list of choice items. In phase 2 (days 15-35): each of the participants was provided with a diet containing 70% of the energy that each participant consumed during the last three days of phase 1. Composition of the diets was determined as described in (Winkels et al. 2011, PMID: 21527169). Blood samples were taken at the end of phase 1, before CR, and at the end of phase 2, after CR.
Growth protocol
Subjects were recruited by publishing advertisements in local newspapers and by sending out general e-mails to an e-mail list of persons who had indicated their interest in participating in studies of our university. Only men were included, because of hormonal fluctuations in young women may influence outcomes. During a screening visit, subjects donated a fasting blood sample and gave written informed consent. In the fasting blood sample, glucose, hemoglobin, and hematocrit were measured. Subjects were excluded based on the following exclusion criteria: body mass index (BMI in kg/m2) less than 20 or higher than 30, adherence to a weight-reduction or medically prescribed diet, dementia (Mini-Mental State Examination score < 21), diabetes, anemia, gastrointestinal disorders, use of drugs known to interfere with energy ballance, or a history of medical or surgical events known to affect the study outcome. The study was approved by the Medical Ethical Committe of Wageningen University.
Extracted molecule
total RNA
Extraction protocol
PBMCs were isolated from whole blood using BD Vacutainer Cell Preparation Tubes according to the manufacturer’s instructions. Total RNA was isolated from PBMC samples using Trizol reagent (Invitrogen, Breda, The Netherlands) and purified using Qiagen RNeasy Micro Kit (Qiagen, Venlo, The Netherlands). RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
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).
Data processing
Expression estimates were calculated applying the robust microarray analysis (RMA) algorithm.
