The fluorescence is measured continuously as the temperature rises and plotted against temperature. A curve is produced, as the fluorescence decreases slightly in the lower temperature range, but decreases much more rapidly at higher temperatures when the melting temperatures (Tm) of specific and nonspecific PCR products are reached. The detection system calculates the first derivatives of these curves, resulting in plots peaking at the respective specific melting temperature of this PCR product (Tm). These Tms may be used to check the specificity of the resulting PCR products. Agarose gel electrophoresis After real-time PCR, the samples were analyzed by 2% agarose gel electrophoresis containing 0.3 mg/ml ethidium bromide at 100 V for 1 h in order to identify the amplicon sizes of the PCR products. As molecular weight marker the GenPilot 50 bp Ladder (QIAGEN) was used. Statistical analysis For the statistical analysis of the expression data the student’s t test was performed and p-values were calculated using the unpaired two-tails method in Excel (Microsoft). Fold changes with p-values,0.05 were considered to be statistically significant, and fold changes with p-values,0.01 to be highly significant. For the P465L DN mouse study results were analysed by two-way ANOVA using SPSS 17.0 (IBM, New York). Data were considered to be significant if there was either an effect of diet or genotype or if a diet*genotype interaction with a p-value,0.05 was observed. For the analysis of the 66 miRNAs we corrected for False Discovery Rate was corrected for using q statistics calculated using R (R Foundation). P values were accepted as valid if the Q statistic was less than P = 0.05. Results Adipocyte-differentiation of murine mesenchymal stem cells For this study, bone marrow-derived murine mesenchymal stem cells from 4 week-old C57BL/6 mice, that have been prepared by depletion of CD45-expressing cells and that have been shown to express CD34, c-kit, sca1 and CD13 [19], were used as the adipogenesis model. Adipogenic differentiation was induced as illustrated in Fig. 1A. Briefly, cells were cultivated until they reached confluency. Then cells were grown for an additional three days to induce growth arrest and subsequently the normal culture medium was changed to adipocyte differentiation medium (day 0). After cultivating the cells for further three days medium was changed to maintenance medium (at day 3) which subsequently was renewed every two days. Samples were taken from subconfluent cell culture (as a reference), at day 0, and 2, 4, 7 and 10 days after induction of adipocyte differentiation. The differentiation progress of two independent cell batches (referred to as passage 5 and passage 8) was monitored by measuring the induction of the adipocyte-differentiation markers adiponectin (Adipoq), CD36 and PPARc by real-time PCR (compare Fig. 1B). All three markers were up regulated in both cell passages upon differentiation with highest expression levels between day 7 and 10 after differentiation. In parallel, adipocyte-differentiation could be detected by visual inspection of the cell cultures. While undifferentiated cells showed a flat and fibroblast-like shape (Fig. 1C), cells became more and more rounded during the differentiation process. In parallel, the formation of multilocular lipid droplets in the cytoplasm could be observed in differentiating adipocytes which became bigger during this process (compare Genome-Wide Profiling of MicroRNAs Fig. 1D). These vesicles could specifically be stained with Oil Red O which was a further proof of the successful adipocyte differentiation (data not shown). Cells from both passages showed slightly different kinetics of adipogenesis; while cells from passage 5 were terminally differentiated at day 7 after induction, cells from passage 8 differentiated slightly slower and reached this stage at day 10 after initiation of adipocyte differentiation. This was reflected both by the expression pattern of adipocyte marker genes as well as by the morphology of the cells. Because of this obvious difference between both cell passages, we included day 10 of passage 8 in the following analyses whereas for passage 5, day 7 was the last time point that was included in the following miRNA expression profiling. Screening of miRNA expression in MSC-derived differentiated adipocytes A PCR-based approach employing miRNA-specific primers for all known mouse microRNAs (583 in miRBase v.12) was used to profile the expression of these molecules during adipogenesis. In an initial screening, we used pools of five RNA samples from passage 5 to get first information about the overall expression of the complete miRNome that could be detected in these cells. For this purpose, we pooled RNA from undifferentiated proliferating MSC (as reference), and from cells of days 0, 2, 4 and 7 after initiation of adipogenesis. This procedure (as depicted in Fig. 2A) allowed us to quickly sort out miRNAs not being expressed in any of the samples irrespective of their differentiation status. By using this pooling approach, we found that 296 of all miRNAs were absent in the pooled samples (CTs of .35), and that 36 miRNAs were only weakly expressed (CT values between 30 and 35). 221 miRNAs showed an average expression level (CTs between 20 and 30) and 30 miRNAs displayed high expression levels with CT values less than 20 (Fig. 2B). The specificity of the amplified PCR- products was assessed by melting curve analysis. Because of the largely uniform nature of the miRNA-specific PCR amplicons (compare Fig. 2C), the expected Tm for true positive PCR products should be 75uC (+/2 2uC). The specificity of most miRNA PCR assays leading to detectable CT values could therefore be confirmed by analyzing their respective melting curves. As shown in Fig. 2D and 2E, specific PCR products with melting temperatures of about 75uC did also posses the expected size of 85 bp demonstrated by agarose gel electrophoresis (a representative result for mir-30a is given). Thus, unspecific PCR products displaying high Tm values could be easily identified by melting curve analysis. Melting temperatures of about 80uC or higher indicate that the PCR product is most likely unspecific. In this rare event, subsequent gel analysis revealed single or multiple PCR products of the wrong size. The two examples shown here are miR-21* displaying a melting curve peak at 79uC and multiple PCR products ranging in size between 80 and 300 bp and miR- 125a-3p exhibiting a Tm of 80uC and one distinct PCR product of about 230 bp (see Fig. 2D and 2E). Such microRNA PCR assays giving rise to PCR products not passing the quality criteria described here were subsequently excluded form further analysis. For appropriate normalization of the miRNA expression values, we intended to identify an endogenous small housekeeper transcript showing low expression variation during adipogenesis. For this purpose, we separately analyzed the expression of the six small RNAs SNORA73A, SNORD25, SCARNA17, RNU6B, RNU5A and RNU1A in all samples taken during the differenti- ation of both MSC passages 5 and 8. These small transcripts are highly suitable candidate RNAs for normalization of miRNA expression levels because they are ubiquitously expressed, have small sequence lengths of 45–200 bp and are, like mature PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21305