Sex: male mouseid: B4_01 diet group: CR diet group (full name): calorie restriction age at sacrifice (months): 28M diet switch at the age of 24m: no tissue: colon strain: C57BL/6J
Treatment protocol
Prior to sacrifice, each mouse was first fasted for 4 hours after which they received an intragastric gavage of 400ul of 0.5% carboxymethyl cellulose, then fasted again for another 6 hours. The animals in CR diet group were fed with half of the portion they normally received 15 min before the first fasting. During the sacrifice, after sedation with a mixture of isoflurane (1.5%), nitrous oxide (70%) and oxygen (30%), tissue was snap-frozen and stored at -80C until further analysis.
Growth protocol
The design of the study and experimental procedures have been described in detail previously (PMID: 25504628). Briefly, male C57BL/6J mice (age: 7 weeks) were purchased from Janvier (Cedex, France) and were housed in pairs of two in the light and temperature (20C)-controlled animal facility of Wageningen University (12-hour light/dark cycle, lights on at 04.00). The mice received standard AIN-93G (Research Diet Services, Wijk bij Duurstede, The Netherlands) for 2 weeks upon arrival. At the age of 9 weeks, mice were individually housed and randomized to different dietary regimens. For the current analyses, we considered mice that were lifelong fed a semi-synthetic control diet consisting of 10E% of fat (C; control), a calorie restriction (CR) diet without malnutrition (30% energy reduction compared to the C diet) or a medium fat (MF) diet consisting of 25E% of fat. All diets, except the CR diet, were fed ad libitum. Mice sacrificed at 6 or 28 months of age were considered in order to differentiate between effects at young and old age. To study the molecular flexibility and dynamics after receiving a strict CR diet for prolonged time, a diet switch was included in this study. A subgroup of mice on CR was switched to the MF diet at the age of 24 months until sacrifice at 28 months.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the scrapings using TRIzol Reagent according to the manufacturer’s instructions (Invitrogen, Breda, The Netherlands). Isolated RNA was purified using RNeasy Micro columns (Qiagen, Venlo, The Netherlands) and total RNA yield (Nanodrop ND-1000, Nanodrop Products, Maarssen, The Netherlands) and RNA integrity (Agilent 2100 Bioanalyzer, Agilent Technologies, Amsterdam, The Netherlands) were assessed. 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
Purified total RNA (100ng per sample) was labeled with the Whole-Transcript Sense Target Assay (Affymetrix, Santa Clara, CA, USA; P/N 900652).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Mouse Gene 1.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.40.1).
