Imprinted miRNA Signature in Male GS Cells vector (Promega, Madison, WI, USA), sequenced in 3730 DNA Analyzer (Applied Biosystems) and analyzed for methylation status using BiQ Analyzer [35]. As control, genomic DNA from mouse sperm and oocytes were also analyzed. Analyses of gene expression Gene expression in GS and maGS cells were analyzed by semi-quantitative RT-PCR or real time quantitative RT-PCR (qRT-PCR) essentially as we described earlier [10,11]. The primer pairs used for PCR amplification are shown in Supplementary Table S2. Statistical analyses The expression levels of genes and miRNAs were compared by ANOVA using SPSS software (SPSS Inc, Chicago, IL, USA). Differences at P#0.05 were considered significant. Results and Discussion We observed that the expression pattern of imprinted miRNAs in mouse sperm was distinct from those of ES or somatic cells (Figure 1B). Sperm showed significantly higher (P,0.001) expression of imprinted and paternally expressed miRNAs (miR- Figure 2. Expressions of miR-296-3p, miR-296-5p, miR-127, miR-127-5p and miR-483 imprinted miRNAs in male germ-line (GS) and multipotent adult germ-line (maGS) stem cells. Sperm and Embryonic stem (ES) cells were used as controls for comparison. All data were normalized to endogenous sno202 RNA as internal controls and calibrated on the STO cells, whose expression was considered one for all genes except for the miR-127 and miR-5p, for which sperm were used as a calibrator and considered one. Values above the bars indicate relative abundance of respective miRNAs. Different alphabet (a, b, c. d) on the bars indicate statistical difference (P,0.01) in gene expression. doi:10.1371/journal.pone.0022481.g002 PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22481