Journal: Molecules and Cells

Article Title: PGC-Enriched miRNAs Control Germ Cell Development

doi: 10.14348/molcells.2015.0146

Figure Lengend Snippet: The expression levels of miRNAs during male germ cell development. (A) Expression profiling of 12 PGC-enriched miRNAs identified by miRNA microarray in developing male germ cells. For each miRNA, the mean-centered log 2 -expression profile (mean = 0) is shown. A hierarchical clustering was done to visualize the similarity in expression patterns of the miRNAs. The colors represent increased (red) and decreased (green) expression relative to the mean expression level. (B) PGC-enriched expression of miR-10b, -18a, -93, -106b, -126-3p, -127, -181a, -181b, and -301 (red) among 12 miRNAs identified from a miRNA microarray analysis were confirmed by miRNA qPCR. (C) Northern blot analysis demonstrates that miR-18a, -93, -106b, and -181a were expressed in PGCs during male germ cell development. In the bottom, Ethidium bromide staining of 28S, 18S, and 5S rRNAs is shown as a loading control. Northern blot assay was performed as described in Materials and Methods. (D) miRNA in situ hybridization assay demonstrated the PGC-enriched expression patterns of miRNAs in PGCs at 12.5 dpc. Arrow, a seminiferous cord; black arrowhead, primordial germ cells; white arrowhead, Sertoli cells.

Article Snippet: A hybridization assay was carried out with Genopal®-MICM miRNA arrays (Mitsubishi Rayon, Japan) overnight, where 180-oligonucleotide DNA probes are installed for detection of mouse miRNAs in 150 μl of hybridization buffer containing 2x SSC, 0.2% SDS, and ∼1 μg of heat-denatured labeled RNAs at 50°C.

Techniques: Expressing, Microarray, Northern Blot, Staining, Control, In Situ Hybridization

Journal: Molecules and Cells

Article Title: PGC-Enriched miRNAs Control Germ Cell Development

doi: 10.14348/molcells.2015.0146

Figure Lengend Snippet: Identification of target genes by PGC-enriched miRNAs and their biological processes. (A) The expression of target genes categorized in C1 and C2 showed a reverse correlation with nine PGC-enriched miRNAs during germ cell development. The auto-scaled log 2 -expression profiles of each target gene (mean = 0 and standard deviation = 1) are shown (gray line). Median expression values in C1 and C2 for each sample are expressed as blue and red lines. (B) The strategy for identifying target genes by nine PGC-enriched miRNAs. 6,511 targets were generated from the three databases using the sequence pairing algorithms. Among them, 127 were selected as target genes having a reverse expression (C1 and C2) with miRNA expression. (C) A heat map showed expression patterns of the 127 target genes. For each gene, the mean-centered log 2 -expression profile (mean = 0) is shown. A hierarchical clustering was done to visualize the similarity in expression patterns of the genes. The colors represent increased (red) and decreased (green) expression relative to the mean expression level. (D) miR-106b and -93 shared target genes due to possession of similar seed sequence. Similarly, miR-181a and -181b shared target genes.

Article Snippet: A hybridization assay was carried out with Genopal®-MICM miRNA arrays (Mitsubishi Rayon, Japan) overnight, where 180-oligonucleotide DNA probes are installed for detection of mouse miRNAs in 150 μl of hybridization buffer containing 2x SSC, 0.2% SDS, and ∼1 μg of heat-denatured labeled RNAs at 50°C.

Techniques: Expressing, Standard Deviation, Generated, Sequencing

Journal: Molecules and Cells

Article Title: PGC-Enriched miRNAs Control Germ Cell Development

doi: 10.14348/molcells.2015.0146

Figure Lengend Snippet: A network model delineates biological processes regulated by PGC-enriched miRNAs. (A) The enrichment analysis results of GOBPs by total genes in C1 and C2 (red bar, total genes) and 127 target genes (blue bar). The red line denotes the cutoff value (P-value = 0.1) of the analysis. (B) A network model shows the relationships between nine PGC-enriched miRNAs and their target genes. Colors and shapes of the nodes represent PGC-enriched miRNAs (green diamonds), target genes in C1 (purple circles) and C2 (red circles). Edge colors were used to distinguish the interactions of individual miRNAs or miRNA families. The nodes were grouped according to their gene ontologies (colored rectangles). Nodes containing the same gene ontologies belong to same group as described in “Materials and Methods”. The genes with multiple gene ontologies were comprised of two or more functional groups. The background colors represent gene ontology groups (ellipsoidal for lower-level gene ontologies and rectangle for upper-level gene ontologies).

Article Snippet: A hybridization assay was carried out with Genopal®-MICM miRNA arrays (Mitsubishi Rayon, Japan) overnight, where 180-oligonucleotide DNA probes are installed for detection of mouse miRNAs in 150 μl of hybridization buffer containing 2x SSC, 0.2% SDS, and ∼1 μg of heat-denatured labeled RNAs at 50°C.

Techniques: Functional Assay

Journal: Molecules and Cells

Article Title: PGC-Enriched miRNAs Control Germ Cell Development

doi: 10.14348/molcells.2015.0146

Figure Lengend Snippet: A heat map represents the relationship between each PGC-enriched miRNA and the twelve biological processes. (A) A heat map shows the contribution score defined by the number of target genes regulated by each miRNA within a gene ontology group divided by the total number of the target genes of the miRNA within the group. (B) RT-PCR analysis confirms expression of the representative target genes during male germ cell development.

Article Snippet: A hybridization assay was carried out with Genopal®-MICM miRNA arrays (Mitsubishi Rayon, Japan) overnight, where 180-oligonucleotide DNA probes are installed for detection of mouse miRNAs in 150 μl of hybridization buffer containing 2x SSC, 0.2% SDS, and ∼1 μg of heat-denatured labeled RNAs at 50°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing