Journal: Journal of Virology

Article Title: MicroRNA gga-miR-130b Suppresses Infectious Bursal Disease Virus Replication via Targeting of the Viral Genome and Cellular Suppressors of Cytokine Signaling 5

doi: 10.1128/JVI.01646-17

Figure Lengend Snippet: gga-miR-130b directly targets the IBDV genome. (A) Diagram of predicted target sites for miR-130b in IBDV genomic RNA. The seed sequence of miR-130b is underlined and was mutated as indicated by the arrow. (B) miR-130b inhibited target gene expression in a dose-dependent manner. DF-1 cells were cotransfected with luciferase reporter vectors containing wild-type (WT) target sites, pRL-TK, and miR-130b at different concentrations. At 48 h posttransfection, cells were lysed and luciferase reporter gene assays were performed to measure luciferase activities. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miR-130b mimics/luciferase activity of cells cotransfected with the reporter plasmid and miRNA controls. (C) A point mutation in the target gene abolished miR-130b-induced suppression of the target gene. DF-1 cells were cotransfected with miR-130b and the WT or mutant luciferase reporter vector. At 48 h posttransfection, a luciferase reporter gene assay was performed to measure luciferase activity. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Article Snippet: Deep sequencing was performed by LC Sciences (Hangzhou, China).

Techniques: Sequencing, Targeted Gene Expression, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis, Reporter Gene Assay

Journal: Journal of Virology

Article Title: MicroRNA gga-miR-130b Suppresses Infectious Bursal Disease Virus Replication via Targeting of the Viral Genome and Cellular Suppressors of Cytokine Signaling 5

doi: 10.1128/JVI.01646-17

Figure Lengend Snippet: The SOCS5 gene is a cellular target of gga-miR-130b. (A) Diagram of predicted target sites for miR-130b in the SOCS5 gene. The seed sequence of miR-130b is underlined and was mutated as indicated by the arrow. (B) Transfection of gga-miR-130b reduced expression of SOCS5. DF-1 cells were cotransfected with miRNAs and luciferase reporter vectors. At 48 h posttransfection, cells were lysed, and a luciferase reporter gene assay was performed to measure SOCS5 expression. The relative level of luciferase activity was calculated as follows: luciferase activity of reporter plasmid-transfected cells or cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. (C) Mutation of the target site abolished the inhibition of SOCS5 by miR-130b. DF-1 cells were cotransfected with miRNA controls or miR-130b mimics or inhibitors and luciferase reporter vectors. At 48 h posttransfection, the assay was performed to measure the luciferase activity. The relative level of luciferase activity was calculated as described above. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001.

Article Snippet: Deep sequencing was performed by LC Sciences (Hangzhou, China).

Techniques: Sequencing, Transfection, Expressing, Luciferase, Reporter Gene Assay, Activity Assay, Plasmid Preparation, Mutagenesis, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells

doi: 10.3390/ijms18081826

Figure Lengend Snippet: Expression profiles of miRNAs in human UTUC tumor and non-tumor samples. Three tumor (T1–T3; light blue to navy blue) and three adjacent normal tissues (N1–N3; orange-yellow-green) were subjected to RNA extraction and NGS-miRNA sequencing through the Illumina MiSeq platform. ( A ) Heat-map clustering analysis was conducted to examine the overall miRNA expression profiles among samples. A line scatterplot was used to present miRNA expression profiles; ( B ) Downregulation of miR-30a-5p in UTUC tumor tissues ( n = 22) compared with adjacent normal controls ( n = 14) by RT-qPCR analysis. The asterisk denotes p < 0.001 using an unpaired t -test.

Article Snippet: The small RNA library construction and deep sequencing was carried out at a Biotechnology Company (Welgene, Taipei, Taiwan).

Techniques: Expressing, RNA Extraction, Sequencing, Quantitative RT-PCR

Journal: Human Molecular Genetics

Article Title: Altered keratinocyte differentiation is an early driver of keratin mutation-based palmoplantar keratoderma

doi: 10.1093/hmg/ddz050

Figure Lengend Snippet: Analysis of global gene expression in Krt16-null footpad lesions and comparison to human PC cases. (A) Volcano plot depicting differentially expressed genes in Krt16-null paw skin lesions relative to WT controls. (B) Top 10 genes differentially regulated in Krt16-null paw skin from established paw lesions compared to WT littermate controls. (C) Validation of downregulated genes identified by microarray analysis by RT-qPCR in Krt16-null paw skin lesions. N = 4 mice/genotype. Error bars are SEM. *P < 0.05, **P < 0.01. (D) Overlap in the significantly changed genes that are upregulated between Krt16-null paw skin lesions and KRT16 human cases. (E) Overlap in the significantly changed genes that are downregulated between Krt16-null paw skin lesions and KRT16 human cases. (F) Overlap in the significantly changed genes that are upregulated between Krt16-null paw skin lesions and KRT6 human cases. (G) Overlap in the significantly changed genes that are downregulated between Krt16-null paw skin lesions and KRT6 human cases. Boxes for (D)–(G) list common genes between mouse and human data sets (listed in alphabetical order).

Article Snippet: Total RNA isolated from paw skin, epidermis and dermis, of 2 month old Krt16 -null (2 males, 2 females) and WT (2 males, 2 females) littermates (see Biochemical and morphological analyses ) was subjected to microarray analysis by the Johns Hopkins Deep Sequencing Microarray Core using the Affymetrix Mouse Transcriptome Array 1.0 GeneChip.

Techniques: Gene Expression, Comparison, Biomarker Discovery, Microarray, Quantitative RT-PCR