Supplementary MaterialsAdditional file 1: Table S1. Additional file 5: Table S4.
May 11, 2019
Supplementary MaterialsAdditional file 1: Table S1. Additional file 5: Table S4. Detailed results of GO terms analysis for gene transcripts targeted by miRNAs (Table S4A showed the GO terms of target genes of the miRNAs that are more abundant in lambs than in adults; Table Clozapine N-oxide manufacturer S4B showed the GO Clozapine N-oxide manufacturer terms of target genes of the miRNAs that are less abundant in lambs than in adults). (XLS 922?kb) 12864_2018_4736_MOESM5_ESM.xls (923K) GUID:?0F5533D5-C445-42C6-9605-913657E1DA9A Additional file 6: Table S5. Signaling pathways revealed by KEGG analysis that involve miRNAs highly expressed in lambs and their target genes (Table S4A showed pathways involving target genes of the miRNAs that are more abundant in lambs than in adults; Table S4B showed pathways involving target genes of the miRNAs that are less abundant in lambs than in adults). (XLS 254?kb) 12864_2018_4736_MOESM6_ESM.xls (254K) GUID:?8724E53E-E50B-4576-BC3C-607200075959 Additional file 7: Table S6. The signaling pathways analysis Clozapine N-oxide manufacturer with target genes that form miRNA-mRNA pairs with differentially expressed miRNAs between the two stages of Tan sheep. (XLS 30?kb) 12864_2018_4736_MOESM7_ESM.xls (30K) GUID:?99CB3100-DDDD-4ACA-9ADC-30AFBC8B2F64 Additional file 8: Table S7. Sequences of primer used for amplification of wild type or mutant KRT83 CDS region. (DOCX 48?kb) 12864_2018_4736_MOESM8_ESM.docx (48K) GUID:?298E27D3-AF5D-4546-8B06-CEA590C285A0 Data Availability StatementAdditional data can be found in supplementary files. Abstract Background Tan sheep is an indigenous Chinese breed well known for its beautiful curly fleece. One prominent breed characteristic of this sheep breed is that the degree of curliness differs markedly between lambs and adults, but the molecular mechanisms regulating the shift are still not well understood. In this study, we identified 49 differentially expressed (DE) microRNAs (miRNAs) between Tan sheep at the two stages through miRNA-seq, and combined the data with that in our earlier Suppression Subtractive Hybridization cDNA (SSH) library study to elucidate the mechanisms underlying curly fleece formation. Results Thirty-six potential miRNA-mRNA target pairs were identified using computational methods, including 25 DE miRNAs and 10 DE genes involved in the MAPK signaling pathway, steroid biosynthesis and metabolic pathways. With the differential expressions between lambs and adults confirmed by qRT-PCR, some miRNAs were already annotated in the genome, but some were novel miRNAs. Inhibition of expression by was confirmed by both gene knockdown with siRNA and overexpression, which was consistent with the miRNAs and targets prediction results. Conclusion Our study represents the comprehensive analysis of mRNA and miRNA in Tan sheep and offers detailed insight into the development of curly fleece as well as the potential mechanisms controlling curly hair formation in humans. Electronic supplementary material The online version of this article (10.1186/s12864-018-4736-4) contains Clozapine N-oxide manufacturer supplementary material, which is available to authorized users. and were significantly higher in lambs than in adults, while levels of and were lower in lambs (Fig.?6). These results are similar to those obtained using miRNA-Seq (Table ?(Table2).2). Although there were some quantitative differences between the two analytical platforms, the overall agreement suggests that the miRNA-Seq data are reproducible and reliable. Open in a separate window Fig. 6 Validation of selected DE miRNAs through bulge-loop RT-qPCR technology. Each bar represents relative expression Clozapine N-oxide manufacturer (mean??SEM) of a miRNAs normalized by U6 small nuclear RNA in lambs (L) or adults (A). *indicates was direct targets of mRNA, we thus used dual luciferase reporter system to test whether could target expression (Fig.?7). As shown by dual-luciferase reporter assay in Hela cell Rabbit Polyclonal to PDGFR alpha line (Fig. ?(Fig.7),7), co-transfection of the Hela cell line with mimic or negative control did not induce any difference in luciferase activity of reporter in cells containing mimic evidently decreased the luciferase activity compared to that in the cells treated by negative control. The result suggested that the previous prediction results were accurate. Open in a separate window Fig. 7 Hela cell culture transfections showing negative relationship between and target Cells were transfected with two plasmids containing wild type (wt) or mutant (mut) CDS region, and then treated with mimic (black) or negative control (grey). Each bar represents relative luciferase activity (mean??SEM) for triplicates of each transfection and treatment combination. ** indicates on expression of at both RNA and protein level, we transfected primary sheep epidermal fiber cells with mimic, and then compared the expression of in cells with that in cells treated by negative control miRNA. As shown in Fig.?8, after the same normalization with as housekeeping genes, qPCR showed no significant difference in mRNA expression between mimic and negative control treated cells, but western blotting showed significant decrease of KRT83 protein in cells treated by mimic compared to those treated by negative control. Open in a separate window Fig. 8 The overexpression of repressed the protein expression but not mRNA expression of KRT83 in primary sheep epidemial.