(RXRs) [14,15,16]. The concerted action of these adipogenic transcription factors ultimately drives the expression of adipocyte specific factors such as enzymes responsible for the synthesis and storage of triglycerides in lipid droplets. [17,18]. Here, we describe the results of a miRNome-wide analysis of primary adipocyte differentiation, conducted by a novel screening strategy. By using pools of RNAs from MSCs at different time points after adipocyte differentiation, we could reduce the complexity of such a real-time PCR based screening approach while keeping the detection sensitivity. Pools of 5 RNAs from undifferentiated MSCs or differentiated cells at four different time points were used for RT-PCR analysis in order to exclude those miRNAs from further tests which were not present at detectable levels in any of the samples. In a second step, detectable miRNA species were investigated in individual RNA samples for their expression patterns during in vitro differentiation of MSC. Regulated miRNA candidates, revealed during the MSC screen, were further studied in vivo using white fat tissue samples from mouse models of genetically or diet induced obesity. Whether the expression of these miRNAs was dependent on PPARc activity in vivo was further analyzed in a mouse model harbouring a dominant-negative PPARc mutation (P465L). Materials and Methods Cell culture All cells were cultured in a 5% CO2 humified atmosphere at 37uC in T25-culture flasks. The culture media were changed every second or third day. Mouse mesenchymal stem cells (MSC) derived from bone marrow of 4 weeks old C57/Bl6 mice were a kind gift from the group of Viktor Wixler, University of M u¨nster. The cells were previously characterized as mesenchymal stem cells by determination of surface marker genes using quantitative RT- PCR and their potential to differentiate into osteogenic, chondrogenic and adipogenic lineages [19]. The MSC were cultivated in a mixture of 60% DMEM and 40% MCDB-201 medium (Sigma-Aldrich) supplemented with 2% FCS, 100 units penicillin/ml, 100 mg/ml streptomycin, 10 ng/ml EGF and 10 ng/ml PDGF (R&D Systems), 1 mg/ml linoleic acid (Sigma- Aldrich), 10 ng/ml leukemia inhibitory factor (LIF, Chemicon International, Inc.), 5 mg/ml insulin-transferrin-selenium mixture (Sigma-Aldrich), 0.1 mM ascorbate-2-phosphate and 1022 mM dexamethasone (DMX) (Sigma-Aldrich). MSC were induced to differentiate at the third day after reaching confluency by addition of differentiation medium (60% DMEM, 40% MCDB-201, 2% FCS, 100 units penicillin/ ml,100 mg/ml streptomycin ,1 mg/ml linoleic acid, 0.1 mM ascorbate-2-phosphate, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 mM DMX and 10 mg/ml insulin-transferrin-selenium mixture). After three days the medium was changed to adipocyte maintenance medium containing the same supplements as the differentiation medium but with hundred fold lower DMX concentration and lacking IBMX. The cells were cultivated for additional 7 days. Total RNA including miRNA was isolated from normal proliferating cells (as reference), as well as from confluent cells at the day of induction (d0), and at two (d2), four (d4), seven (d7) and ten (d10) days after induction using miRNeasy Mini Kit according to the manufacturers protocol (QIAGEN). For verifi- cation of the results, cells from two passages (passages number 5 and 8) were analyzed independently. For visualisation of the differentiated adipocytes, the cells were stained with Oil Red O by washing twice with PBS and fixing with 4% paraformaldehyde in PBS at 4uC over night. The solution was removed and the fixed cells were washed with 60% isopropanol. After drying, cells were Genome-Wide Profiling of MicroRNAs stained in fresh Oil Red O solution (60% Oil Red O stock, consisting of 0.35% Oil Red O in isopropanol, and 40% H2O) for at least one hour at room temperature. Subsequently, the cells were washed several times with water. NIH/3T3-cells (American Type Culture Collection, ATCC) were cultivated in DMEM (Gibco) supplemented with 10% FCS (BioChrom), 100 units penicillin/ml,100 mg/ml streptomycin and non-essential amino acids (Invitrogen). After reaching confluency (k0) the cells were cultivated for further three days (k1–k3) and total RNA was isolated at every time point using miRNeasy Mini Kit according to the manufacturer’s protocol (QIAGEN). Mouse studies Unless stated otherwise, mice were housed in a temperature- controlled room (22uC) with a 12-hour light/dark cycle and had free access to food and water. All animal protocols were approved by the UK Home Office, licence number 80/2098 and the University of Cambridge. The first model consisted of leptin- deficient ob/ob mice on a C57BL/6 genetic background. ob/ob mice were fed a standard laboratory chow diet (10% calories from fat) and were compared to littermate wt control animals. The second group of mice consisted of animals exposed to diet-induced obesity. Mice on a mixed C57BL/6J/SV129 genetic background were fed a high-fat diet (HFD) for 10 weeks from weaning (45% calories from fat) and compared to littermate controls fed a chow diet from weaning. The final experimental group was a mouse model harbouring a dominant-negative PPARc mutation (P465L) fed either a chow diet or HFD. Leptin-deficient ob/ob mice and litter mate controls were purchased from Harlan (Harlan, UK). Animals for the HFD studies and P465L DN mice were bred in- house at Cambridge University. For every model the epididymal fat pads of five 10–13 weeks old male mice were harvested, frozen in liquid nitrogen and stored at 280uC. Total RNA including miRNAs of all tissue samples were extracted using miRNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol and the TissueRuptor (QIAGEN) for homogenisation. Quantitative real-time PCR For mRNA analyses 100 ng total RNA were reverse-transcribed by Omniscript RT Kit (QIAGEN). For real-time PCR amplifica- tion QuantiTect SYBR Green PCR Kit and QuantiTect Primer Assays were used according to manufacturer’s protocol (QIA- GEN). GAPDH was used as endogenous control. For miRNA analyzes 100 ng total RNA were reverse-tran- scribed and amplified in real-time PCR using miScript-System including miScript RT-Kit, miScript SYBR-Green PCR-Kit and miScript Primer Assay miRBase v12 (QIAGEN) according to the manufacturer’s protocol. For internal control the expression of the small nuclear RNAs RNU1A, RNU6B and RNU5A as well as the expression of the small nucleolar RNAs SNORA73A, SNORD25, and SCARNA17 was determined. All PCR reactions were performed in triplicate in 384-well plates and measured by ABI 7900HT (Applied Biosystems). Mean values and standard deviations were calculated and fold changes were determined using the DDCT method. miRNAs displaying a fold change .2 or ,0.5 during adipogenesis of mesenchymal stem cells or a fold change .1.5 or ,0.66 in the in vivo obesity models in combination with p-values,0.05 were regarded as regulated. During the melting curve analysis after completion of the PCR amplification cycles, the temperature is increased slowly from 65uC to 95uC. At low temperatures all PCR products are double stranded and SYBR Green intercalates into these molecules leading to a high fluorescence signal. Whereas at high temperatures, PCR products are denatured, resulting in rapidly decreasing fluorescence signals. PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21305