title: MicroRNA expression differences in adipose tissue between high and low fat diet mice

status: Public on Nov 30 2011

city: Nicosia

first: Apostolos

last: Zaravinos

address: Kallipoleos 75

institute: University of Cyprus

summary: Total RNA was extracted from adipose tissue of high (full) fat diet and standard fat diet mice.

overall_design: Adipose tissue was taken from 16 mice in total. Eight mice were fed a standard diet (SD; control) (10 kcal% fat) and the other eight were fed a high-fat diet (HFD; test) (60 kcal% fat; Research Diets, New Brunswick, NJ) for 5 months. Total RNA was isolated from pooled White Adipose Tissue from SD- or HFD-fed mice using guanidinium thiocyanate. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. One µg total RNA from sample and reference were labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labeling kit (Exiqon, Denmark) following the procedure described by the manufacturer. The Hy3™-labeled samples and the Hy5™-labeled sample were mixed pair-wise and hybridized to the miRCURY™ LNA array version 10.0 (Exiqon, Denmark), which contains capture probes targeting all miRNAs for all species registered in the miRBASE version 11.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm.

first: Dionysis

last: Chartoumpekis