Transcriptional profiling of rat liver after a three days caloric restriction or 3 days starvation. In the present study, we wanted to investigate the expression of multiple genes linked to pathways of energy metabolism. We developed a metabolism dedicated microarray tool: the Mitoligo. This microarray gathers genes linked to the main functions of energy metabolism, such as glycolysis or fatty acid oxidation. This biochip has also been designed to study the expression of genes encoding for all the subunits of proteins constituting the ETC. Eventually, genes coding for protein linked to ROS scavenging systems can also be studied using the Mitoligo. Keywords: Nutritional intervention Overall design: The present investigation was performed according to the guiding principles of the French department of Animal and Environmental protection for the care and use of animals and in accordance with the American Physiological Society “Guiding Principles for Research Involving Animals and Human Beings”. Twenty-four male Sprague-Dawley rats, born and bred in our animal facilities, were housed in individual cages at 12 weeks of age (370-420g). Animals were provided with water ad libitum and a standard diet (U.A.R A04) consisting (by weight) of 16% protein, 3% fat, 60% carbohydrate and 21% water, fiber, vitamins and minerals. The metabolizable energy content was 2.9 kcal/g. Rats were randomly separated into 3 groups as follows: control fed (CON) group (n=6), the calorie restricted (CR) group and the 100% calorie restricted (fasted) group. The rats in the CON group consumed the standard diet ad libitum while the other rats were food-restricted and received 25 and 50% (CR group) of the food given to the control group. The rats in the fested group were totally deprivated of feeding. The experimental design was conducted for 3 days. On the 4th day, the animals were killed by decapitation. Liver was removed rapidly and weighed. Some tissue samples were immediately used for respiration and potential membrane measurements and the remainder were frozen in liquid nitrogen and stored at -80°C in order to measure enzyme activity levels and gene expression levels. Total RNA was isolated from tissue samples using a standard guanidium isothiocyanate protocol (TRIzol reagent, Invitrogen). RNA integrity was determined using a BioAnalyzer 2100 (Agilent Technologies, Waldbronn, Germany). Cy3- and Cy5-labelled (amplified) aRNA was prepared using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion, 1753). The reference sample consisted of a pool of an equal amount of aRNA obtained after the amplification of tRNA from each non-restricted control rat and was labelled with Cy5 (Amersham). For each of different rat within each group (including control group), two replicate tRNA extractions were prepared using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion, 1753) and labeled individually with Cy3 (Amersham). Each Cy3-labeled sample was mixed with an equal amount of the Cy5- labeled reference sample, pre-incubated with yeast tRNA and polyA RNA, and hybridized to the microarrays. Microarrays were prepared in-house (Plateforme Puces à ADN-Ouest Génopole, Nantes, France) using 50-mer oligonucléotides probes. The probes were diluted in spotting buffer A (25 µM in buffer A 1x, Ocimum) and spotted onto epoxysilane-coated glass slides (Slide E, Schott nexterion) using the Lucidea Array Spotter from Amersham. Immediately after spotting, the slides were incubated at 42°C and 55% humidity for 12 h. Slides were washed once in 0.2% SDS and twice in H2O and then incubated for 20 minutes in H2O at 50°C, dried by centrifugation (1000 rpm, 3 minutes, 20°C), and stored at 20°C. The 982 genes that were represented on the microarray were selected for involvement in mitochondrial metabolism. 350 genes are specific for rat while 600 genes are specific of human mRNA. Each gene was spotted in triplicate on the microarrays. Hybridized arrays were scanned at 10 µm/pixel resolution by fluorescence confocal microscopy (Scanarray 3000, GSI-Lumonics). Signal intensities were extracted with Genepix Pro 5.1.0.19 image analysis software (Axon Instruments, USA). Data were retained based on concordance between the measurements of different types of replicates: biological replicates, technical replicates and combined biological and technical replicates. Microarray raw data were analyzed using Genespring GX 7.0 (Agilent technologies). Data were normalized using Lowess normalization. Normalized data were compared using ANOVA followed by tukey or student t-test.