48 hours Mesenteric adipose tissue. Control group (0.2 ml sunflower oil gavage).
Treatment protocol
Female Wistar rats (Harlan-Interfauna Ibérica, Sant Feliu de Codines, Spain) weighing initially 220-230 g were housed in individual cages under a light cycle (on from 08.00 to 20.00) and in a temperature-controlled environment (20-22ºC). Food (standard rat chow pellets, from Panlab, Barcelona, Spain) (available energy: 13.6 MJ/kg) and water were provided ad libitum. The rats were given a daily oral gavage of 10 μmol/kg/day of oleoyl-estrone (OED, Barcelona, Spain) in 0.2 ml of sunflower oil during two days, by means of stomach cannulae. Controls received only the oil gavage.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from adipose mesenteric tissue using Tripure isolation kit (Roche). RNA concentration and quality were measured by spectrophotometric analysis at 260 nm and 280 nm using Nanodrop equipment (Wilmington DE, USA). RNA integrity was assessed by electrophoresis in agarose gel.
Label
33P
Label protocol
Radiolabeled cDNA probes were prepared from 5 µg of total RNA. Briefly, the RNA was incubated for 2 min at 70ºC followed by 2 min at 50ºC with 1 µl of the primer mix, containing the 1168 primers for the genes presented in the array. The RT reaction was carried out using 100U MMLV-RT, 40U RNAsin (Promega, Madison WI, USA) and 35 µCi [-33P] dATP (Amersham Biosciences, Piscataway NJ, UK) for 25 min at 50ºC. Labeled cDNA was purified from unincorporated nucleotide with Atlas NucleoSpin® Extraction Kit (Clontech).
Hybridization protocol
Nylon membranes were pre-hybridized in ExpressHybTM (Clontech) with 100 µg/ml DNA salmon sperm for 30 min in an oven at 68ºC. Then, the 33P-labeled probe was added and hybridization continued overnight at the same conditions. Afterwards, membranes were washed lowering the astringency progressively to 0.1 X SSC, 0.5 % SDS at 68ºC and placed in contact with europium screens (Kodak, Rochester NY, USA) for 15 days.
Scan protocol
The screens were scanned with a Storm 840 phosphorimager (Molecular Dynamics, Sunnyvale CA, USA).
Description
Array data analyses were conducted via a parametric comparison using all available error estimates as a filter based on variances calculated by the Cross-gene Error Model in GeneSpring 6.1 software (Silicon Genetics, Redwood City CA, USA). The Cross-gene Error Model performs a variance components analysis for the accurate comparison of mean expression levels between experimental conditions. In this model, separate estimates for two different kinds of random variation are used to estimate the variability in gene expression measurements: i) measurement variation: this comprises the lowest level of variation, corresponding to the variation of gene measurement of a single chip based on the actual value that would be obtained from a perfect measurement of the gene expression level for that sample, and ii) sample-to-sample variation: the variation between samples under the same condition reflecting biological or sampling variability.
Data processing
Image analysis and quantification were carried out with Atlas Image 2.01 software (Clontech). After grid assignment, the adjusted intensity for each gene was calculated by subtracting the background. This value was used as the input for the GeneSpring 6.1 program (Agilent, Palo Alto CA, USA), that allows normalization of data analysis from different experiments, the generation of restriction lists, and the functional classification of the differentially expressed genes.
