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Whole transcriptome and miRNAome analysis of porcine muscle tissue in the terms of muscle growth and development

ABSTRACT: Purpose: The aim of present research was to identify genes potentially related with muscle growth in pig Methods: RNA-seq analysis was performed on longissimus lumborum samples collected from 13 pigs representing two sire-line breeds – Pietrain and Hampshire. Base on dissection data, pigs were selected from a larger population in terms of weight of loin muscle to obtain the most extreme groups in each breed The cDNA libraries were constructed form 300 ng of total RNA with the use TruSeq RNA Sample Prep Kit v2 kit (Illumina) according to the protocol. The quantification of obtained libraries was performed on Qubit 2.0 (Invitrogen, Life Technologies) and TapeStation 2200 (D1000 ScreenTape; Agilent). The RNA-seq was performed in 101 single-end cycles on HiScanSQ platform (Illumina) with the use of ruSeq SR Cluster Kit v3- CBOT-HS and TruSeq SBS Kit v 3 - HS (Illumina). Each library was sequences in 4 technical replications. The DEGs were detected using DESEq2 software and validation was performed by qPCR. Results: RNA-seq approach allowed to identify 627 and 416 DEGs between pig groups with different loin weight (pvalue <0.05; fold change≤1.5) for Pietrain and Hampshire; respectively. Next, the comparison of obtained genes sets showed 43 genes identified in both breeds. The Gene Ontology analysis (Panther software) confirmed that detected genes were involved in regulation of metabolic process (18 genes), developmental growth or skeletal system development (PCOLCE; BBS2; TBX2; RBBP6; ATAF5). According to David software these genes belong to PI3K-Akt signaling, Focal adhesion and ECM-receptor interaction pathways. Conclusions: Obtained results allow to propose the panel of genes, which can be related with porcine muscle growth and development. Such analysis can be the basis of future research in terms of identification of candidate genes related with important production traits in pigs.

ORGANISM(S): Sus scrofa

PROVIDER: GSE107207 | GEO | 2017/12/01

SECONDARY ACCESSION(S): PRJNA419312

REPOSITORIES: GEO

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Project description:Purpose: The aim of present research was to identify miRNAs potentially related with muscle growth in pig Methods: miRNA-seq analysis was performed on longissimus lumborum samples collected from 13 pigs representing two sire-line breeds – Pietrain and Hampshire. Based on dissection data, pigs were selected from a larger population in terms of weight of loin muscle to obtain the most extreme groups in each breed The miRNA libraries were constructed form total RNA with the use Neb Next Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) according to the protocol. The quantification of obtained libraries was performed on a Qubit 2.0 spectrophotometer (Invitrogen, Life Technologies) and a TapeStation 2200 instrument (D1000 ScreenTape; Agilent). The miRNA-seq was performed in 36 single-end cycles on HiScanSQ platform (Illumina) with the use of TruSeq SR Cluster Kit v3- CBOT-HS and TruSeq SBS Kit v 3 - HS (Illumina). Differentially expressed microRNAs were detected using the DESeq2 software while validation was performed by qPCR. Results: miRNA-seq approach allowed to identify 196 known and 149 potentially novel miRNAs in both breeds. The comparison of miRNA profiles between pig groups with different loin weight showed differential expression of 57 microRNAs for Hampshire pigs and 34 miRNAs for Pietrain pigs. Furthermore, 18 common microRNAs with differential expression were identified for both breeds tested. The Gene Ontology analysis (DIANA mirPath v.3 tool) confirmed that detected genes were involved in regulation of cell cycle, fatty acid biosynthesis, regulation of actin cytoskeleton, lysine degradation and ubiquitin mediated proteolysis. According to the KEGG Database, these DE miRNAs enrich the following pathways, i.a.: the Hippo signaling pathway, TGF-beta signaling pathway and PI3K-Akt signaling pathway. Conclusions: Obtained results allow to propose the panel of miRNAs, which can be related with porcine muscle growth and development. Such analysis can be the basis of future research in terms of identification of candidate miRNAs related with important production traits in pigs.

Project description:Purpose: The aim of present research was to identify miRNAs potentially related with fattness traits in pigs Methods: miRNA-seq analysis was performed on subcutaneous fat samples collected from 22 pigs representing two sire-line breeds – Pietrain and Hampshire and one dam-line – Large White. Based on dissection data, pigs were selected from a larger population in terms of fatness traits to obtain the most extreme groups in each breed. The cDNA libraries were constructed form 300 ng of total RNA with the use NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, United States) according to the protocol. The quantification of obtained libraries was performed on Qubit 2.0 (Invitrogen, Life Technologies) and TapeStation 2200 (D1000 ScreenTape; Agilent). The RNA-seq was performed in 36 single-end cycles on HiScanSQ platform (Illumina) with the use of ruSeq SR Cluster Kit v3- CBOT-HS and TruSeq SBS Kit v 3 - HS (Illumina). Each library was sequences in 4 technical replications. The DEGs were detected using DESEq2 software and validation was performed by qPCR. Results: RNA-seq approach allowed to identify miRNAs differentially expressed between pigs with various fatness traits in each analyzed breeds: 64 for Pietrain pigs; 41 for Hampshire and 46 for Large White pigs (pvalue <0.05; fold change≤1.5). The comparison of obtained miRNAs sets showed 13 miRNAs identified in all three breeds. The Gene Ontology analysis (mirPath v.3.0 DIANA Tools web with DIANA—TarBase v7.0 as a reference) confirmed that detected miRNAs were involved in fatty acid biosynthesis (pvalue <1e-325); fatty acid metabolism (pvalue 6.661338e-16); ECM-receptor interaction (pvalue <1e-325). The most important targeted genes regulated by identified miRNAs were: fatty acid synthase – FASN; Acetyl-CoA carboxylase 1 – ACACA; Malonyl CoA-acyl carrier protein transacylase – MCAT and acetyl-CoA acyltransferase 1 and 2 (ACAA1 and ACAA2 genes). Conclusions: Obtained results allow to propose the panel of miRNAs, which can be related with fatness in pig. Such analysis can be the basis of future research in terms of identification of molecular mechanisms related with adipogenesis and fatness traits in pigs.

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Whole transcriptome and miRNAome analysis of porcine muscle tissue in the terms of muscle growth and development

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