detection of lowly expressed genes [20], pooling of several RNA preparations is not recommended. In direct comparison, the sensitivity of a PCR-based detection with a ‘‘Limit of Detection’’ of ten copies is 1000-times higher than what could be achieved with microarrays [20] [22]. The specificity of the hits of such a microarray analysis could not be easily verified. This in particular is an important aspect to consider when thinking of the detection limitations introduced by the short length of the target structures (mature miRNAs with a length of 21–23 base-pairs). In contrast to current mRNA expression arrays, where multiple target-specific probe sets per gene are employed to guarantee specificity, such a redundant design is not feasible for miRNA microarrays. miRNA expression profiling of mesenchymal stem cell derived adipocytes The murine bone-marrow-derived mesenchymal stem cells used in this study have the potential to differentiate into adipocytes. Accumulation of lipid droplets in the cytoplasm of differentiating cells could be easily used to estimate the percentage of fat cells in the culture. Such stem cell derived adipocytes are a relevant model for white fat tissue differentiation and may help to better understand the underlying differentiation mechanisms and to find potential therapeutic targets for obesity or metabolic diseases. Among the 66 miRNAs found in our study to be differentially regulated in MSC-derived adipocytes, miR-103/107 and miR-143 have already been found to play a crucial role in adipocyte differentiation [23,24]. The direct involvement of miR-143 in adipocyte differentiation has been studied by using antisense oligonucleotides in cultured human pre-adipocytes. In this model, blocking of miR-143 reduced the expression of adipocyte differentiation markers by 50% [25]. Opposite effects have been observed after ectopical expression of miR-143 in 3T3-L1 cells which led to the up-regulation of adipocyte marker genes [24]. It has also been reported that elevated expression levels of miR-103 accelerate adipocyte differentiation upon treatment with differen- tiation factors [24]. The terminal differentiation of mesenchymal stem cells into mature adipocytes in vitro is a process involving two major steps: firstly, cells need to be growth arrested, and secondly the treatment with differentiating agents induces the transformation into adipo- cytes (reviewed in [17]). In order to be able to distinguish miRNA expression changes that were rather related to adipocyte differen- tiation from those that occurred as a result of growth arrest alone, we analyzed the expression of the 66 candidate miRNAs in growth arrested mouse fibroblast cell line NIH/3T3, which does not have the potential to differentiate into adipocytes. Comparing growth arrested cells to normally dividing control cells, the expression of 8 of the 66 analyzed miRNAs was affected by growth arrest in NIH/ 3T3 cells (fold-changes .+/22). Among those, were miR-7a, miR- 7b, miR-146b, miR-199a and miR-199b* that were regulated at very early time-points after induction of MSCs and show the same direction of up- or down-regulation in the NIH/3T3 model. Comparing our data with those obtained with the 3T3-L1 adipogenesis model used by Xie and coworkers further supports the assumption, that these miRNAs may not have a direct role in the adipogenic differentiation process [24]. Only miR-146b is found to be moderately up-regulated, and none of the other seven miRNAs is significantly changed in this study. A direct involvement of miR- 146b in adipogenesis has not yet been described to our knowledge. Recently it has been shown that miR-146b expression could suppress breast cancer metastasis [26], supporting our finding of its involvement in growth regulation rather than adipogenesis. Besides the regulation of miR-103/107 and miR-143, we could also confirm the up-regulation of miR-422b, miR-148a, miR-30c, Genome-Wide Profiling of MicroRNAs and miR30a-5p found in differentiating 3T3-L1 cells by Xie and colleagues [24]. Nine of the miRNAs we found to be regulated have also been described by Esau and co-workers in human white adipocytes [25]. Although differentiated adipocytes have been compared to pre-adipocytes in this study, the overlap was high. Besides miR-103/107 and miR-143, miR-30c was also upregulated. These three miRNAs were the only ones that were consistently upregulated in our analysis as well as in the work of Esau et al. (2004) as well as in Xie et al. (2009). In general, the number of significantly regulated miRNAs we found differentially expressed was higher compared to the other two studies [24]. One obvious explanation is the number of miRNAs that were investigated in the individual studies. Esau and coworkers analyzed differential expression of 254 human and mouse miRNAs and Xie et al. (2009) used gene chip arrays based on miRBase version 9 for the analysis of 375 miRNAs in differentiating 3T3-L1 cells, We have anlaysed the expression of all mouse miRNAs published in miRBase 12, a total of 583 miRNA sequences. miRNA expression in adipose tissue from obese mice Studying the miRNA regulation in different in vivo models can help to strengthen the biological relevance of the findings received from the in vitro differentiation model employed here. In the present study, we have used ob/ob mice as a genetic model for obesity. As a second model, we analyzed fat tissue from mice with diet-induced obesity. either having a normal genetic background or harboring a dominant negative mutation of PPARc. Comparing white fat tissue from obese leptin-deficient ob/ob- mice with tissue from normally fed wild type mice revealed that most of the upregulated miRNAs in the differentiated MSC model were downregulated in fat pads from these obese mice and vice versa. Only a small number (3/20) of microRNAs were consistently regulated in both model systems. Similar observations have been made earlier for adipogenic genes that are normally up-regulated during adipogenesis, but show reduced expression in epididymal fat pads of ob/ob mice [27–29]. The reverse regulation of miRNA expression in white fat tissue of ob/ob-mice compared to differentiated 3T3-L1 adipocytes has also been reported earlier [24]. In this study, miRNAs being regulated in white fat of ob/ob- mice are similarly regulated in TNFa treated differentiated 3T3-L1 adipocytes. As TNFa treatment may induce insulin resistance by reactivating the expression of preadipocyte genes in vitro [30] [24] this may be the reason why the majority of miRNAs found in our study are oppositely regulated in differentiated, insulin-sensitive adipocytes and insulin resistant ob/ob mice. Alternatively, mature adipose tissue is quite different in terms of its make up to a simple adipocyte culture model. Adipose tissue contains multiple cell types including adipocytes, preadipocytes, stem cells, endothelial cells, macrophages and multiple other immune cells. The differential regulation we observed between the MSC culture system and the effect of obesity on adipose tissue in vivo may reflect the regulation of miRNAs in non-adipocyte or preadipocyte cell types. Using our second mouse model of obesity, the influence of a high fat diet on miRNA expression could be analyzed either physiologically or under conditions when PPARc activity was reduced. By comparing lean PPARc wt animals on a chow diet with obese littermates receiving a high fat diet, normally fed PPARc DN and PPARc DN mice with diet-induced obesity, we were able to differentiate effects only induced by diet from those effects observed as a consequence of reduced PPARc activity and effects due to an interaction between diet and genotype. Interestingly, only four of the 21 regulated miRNAs (miR-199a, miR-455, miR-592, and miR-1192) were solely dependent on the genotype; independent of diet, the expression of miR-199a, miR- PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e21305