Genome-Wide Profiling of MicroRNAs Figure 1. Differentiation of mesenchymal stem cells to adipocytes. A: Protocol for the differentiation of mesenchymal stem cells to adipocytes. B: Real-time PCR-based expression analysis of the adipocyte marker genes adiponectin (Adipoq), CD36 and PPARc during adipogenesis of murine mesenchymal stem cells. For verification two cell passages (ip 5 and ip 8) were independently differentiated and analyzed. Data were normalized by GAPDH expression and plotted as fold change (log10 scale) relative to the expression levels of the undifferentiated subconfluent reference cells. C, D: Phenotypical change from fibroblast-like murine mesenchymal stem cells (C, 1006 magnification) to round-shaped adipocytes (D, 406 magnification). doi:10.1371/journal.pone.0021305.g001 miRNAs, not polyadenylated. The arithmetic mean CT and standard deviation of all time-course samples were determined for both cell passages (Fig. 2F shows the standard deviation of the CT -values as a measure of the expression variation during the differentiation time course). The small non-coding RNAs showing the smallest variation of expression in both cell passages were snoRNA73A and RNU1A. We selected SNORA73A exhibiting a standard deviation of 0.12 and 0.18 respectively and not RNU1A because the latter displayed less consistent amplification plots (data not shown). The expression patterns of the 287 miRNAs, that were detectably expressed in the pooled samples, were reanalyzed in both cell passages and for each individual sample of the MSC differentiation time course. We found a total number of 66 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21305