Genome-Wide Profiling of MicroRNAs Figure 4. Analysis of growth arrested NIH/3T3-cells to identify miRNAs regulated due to quiescence of cells. A: Cell numbers of two passages of NIH/3T3-cells were counted at subconfluence, at the day of confluency and at the following three days to identify the time-point of complete proliferation arrest. B: Expression kinetics of miRNAs during proliferation arrest of NIH/3T3 cells were determined by RT-PCR. Data were normalized to SNORA73A and plotted as relative expression changes compared to subconfluent reference cells. doi:10.1371/journal.pone.0021305.g004 In vivo miRNA expression in adipose tissue from obese mice To gain further insight into the role of the microRNAs that have been found to be differentially expressed during adipocyte differentiation of mesenchymal stem cells, we investigated the transcription patterns of these miRNAs in white adipose tissue derived from several in vivo model systems. As a model for genetic obesity we used leptin-deficient ob/ob mice that were compared with corresponding wt littermates. In the second model, obesity of the animals was induced by high fat diet (as described in materials and methods) and these mice were compared with littermates fed a chow diet. The degree of obesity is displayed by the epididymal fat pad weight of the corresponding animals (Fig. 5A). In ob/ob-mice, the epididymal fat pad weight was 3.24 g, more than 15 fold greater than that of the corresponding control animals (n = 8 per group). In the case of the diet induced obesity model, the fat pad weight of HFD animals was 1.24 g, 4 fold greater than that of the control group (n = 6 per group). By comparing white fat tissue of leptin-deficient ob/ob mice with the corresponding tissue from wild type mice with the same genetic background (C57BL/6), we found 20 miRNAs being differentially expressed (cutoff: 1.5-fold up regulation and 0.66 fold down regulation; p,0.05). Of these 20 miRNAs three were up-regulated in ob/ob-mice whereas the other 17 were down-regulated compared to wt animals. Student’s PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e21305