Genome-Wide Profiling of MicroRNAs Figure 2. Real-time PCR based profiling of miRNAs being differentially regulated during the course of MSC differentiation to adipocytes. A: RT-PCR based pooling strategy to identify all miRNAs being expressed in any of the samples taken during the differentiation time- course. B: Summary of the expression levels of 583 miRNAs analyzed with the pooling strategy. C: Schematic overview of a typical miRNA PCR amplicon produced by SYBR Green based miScript PCR assays. PCR products are typically around 85 bp long and of these 20 bp resemble the variable miRNA sequence while the remaining 60 bases contain the universal PCR primer binding site introduced during reverse transcription. D–E: Melting curve analysis (D) in combination with agarose gel electrophoresis (E), Lane 1: miR-21*, lane 2: miR-125a-3p, lane 3: miR-30a PCR-products, ‘‘L’’: GelPilot 50 bp Ladder revealed the specificity of miRNA PCR products. F: Identification of the most suitable housekeeping gene for the normalization of the miRNA expression data. For this purpose the expression of six small RNAs was analyzed at all time points and in both cell passages. To identify the small RNA showing the lowest variation over the whole sample set the mean values and standard deviations of all time points were calculated for every gene and cell passage. The table shows the standard deviations of these means. doi:10.1371/journal.pone.0021305.g002 miRNAs showing expression changes comparing undifferentiated dividing cells with differentiated adipocytes in both cell replicates (including days d0, d2, d4, and d7 in the case of passage 5 and d0, d2, d4, d7 and d10 for passage 8) with fold changes higher than 2 or less than 0.5. The individual expression data for each of the 66 differentially regulated miRNAs is listed in supporting table S1. Of these, 56 miRNAs were upregulated during adipogenesis whereas ten of them showed reduced expression in differentiated cells. Members of 5 miRNA families were coordinately up regulated during adipogenesis, namely let-7a, let-7c, let7-e, let-7g, and let-7i; members of the miR-29 family miR-29a, miR-29b, and miR-29c; miR-30a_1, miR-30b, miR- 30c, miR-30d, and miR-30e; miR-181a, and miR-181b; miR- 199a, miR-199a-3p, miR199-b*. Besides these, miR103/107 and miR-143, were also found to be upregulated in the adipogenesis model analyzed here. Fig. 3 is showing the representative expression kinetics for four upregulated and three down regulated miRNAs in both cell passages. Analysis of miRNAs regulated early after induction of adipogenesis The differentiation of mesenchymal stem cells into adipocytes requires two steps, cell cycle arrest and treatment with differentiating agents (IBMX, DMX). To identify the miRNAs that have been up- or down-regulated because of the growth arrest of the MSCs, confluent non-dividing NIH/3T3 mouse fibroblasts, which are not capable of differentiating into adipocytes, were tested as well. Cell counting of two individual cell passages (passage 39 and passage 43) revealed that cells stopped dividing one day after reaching confluency (Fig. 4A). The expression of the 66 microRNAs being regulated during adipogenesis of mesenchymal stem cells was also determined from growth arrested NIH/3T3 cells at four different time points (k0–k3) and compared to a reference of subconfluent dividing NIH/3T3 cells. In MSCs, some miRNAs were differentially regulated in both cell passages early after IBMX, DMX treatment with fold-changes .2 at day 2 after induction (e.g. miR-455; Fig. 3). In the confluent NIH/3T3 cells, only a small number of miRNAs was up- or down-regulated more than two- fold, five miRNAs showed induction of expression in both passages, three miRNAs were down-regulated at confluency (Fig. 4B). Five of these eight miRNAs (miR-199a, miR-7, miR- 146b, miR-7b, and miR-199b*) were similarly regulated early during the adipogenesis process of MSCs, indicating that their regulation was due to cell cycle arrest rather than adipogenesis. Whereas miR-411 and miR-703 were regulated late during the adipocyte differentiation process and miR-129-3p was up- regulated in growth-arrested NIH/3T3 cells but down-regulated in MSCs, indicating that these three miRNAs play different roles in NIH/3T3 cells and differentiating MSCs. Figure 3. Expression kinetics of miRNAs being differentially regulated during differentiation of MSC to adipocytes. The expression of miRNAs was analyzed by real-time PCR individually at all time points during the differentiation course and for two independent cell passages (5 and 8) to identify miRNAs being regulated during adipogenesis of murine mesenchymal stem cells. Data were normalized to SNORA73A and plotted as relative miRNA expression levels compared to the level of undifferentiated subconfluent reference cells. doi:10.1371/journal.pone.0021305.g003 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21305