scholarly journals Comprehensive MicroRNA Expression Profile of the Mammary Gland in Lactating Dairy Cows With Extremely Different Milk Protein and Fat Percentages

2020 ◽  
Vol 11 ◽  
Author(s):  
Xiaogang Cui ◽  
Shengli Zhang ◽  
Qin Zhang ◽  
Xiangyu Guo ◽  
Changxin Wu ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Rna Sequencing ◽  
Differentially Expressed Genes ◽  
Small Rna ◽  
Milk Protein ◽  
Milk Composition ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Rna Seq

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

scholarly journals Integrated Small RNA Sequencing, Transcriptome, and GWAS Data Reveal miRNA Regulation in Response to Milk Protein Traits in Chinese Holstein Cattle

2021 ◽  
Author(s):  
Wentao Cai ◽  
Cong Li ◽  
Junya Li ◽  
Jiuzhou Song ◽  
Shengli Zhang
Keyword(s):  
Protein Synthesis ◽  
Rna Sequencing ◽  
Small Rna ◽  
Protein Concentration ◽  
Milk Protein ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Production Traits ◽  
Economic Traits ◽  
Chinese Holstein

Abstract Background: Milk protein is one of the most important economic traits in the milk industry. Our previous study has revealed some functional genes responsible for milk protein synthesis in mammals. Yet, the miRNA-mediated gene regulatory network for the synthesis of milk protein in mammary is poorly understood. Results: 12 samples from Chinese Holstein Cows with three too high and three low phenotypic values for milk protein percentage in lactation and non-lactating were examined through deep small RNA sequencing. By bioinformatics analysis, we characterized 387 known and 212 novel miRNAs in the mammary gland. Differentially expressed analysis detected 28 miRNAs in lactation and 52 miRNAs in the non-lactating period with a highly significant correlation with milk protein concentration. Target prediction and correlation analysis identified some key miRNAs and their targets potentially involved in the synthesis of milk protein. Using genome-wide association signal (GWAS) enrichment analysis among five milk production traits, we found the differentially expressed targets were significantly related to milk protein traits.Conclusions: This integrated study on the transcriptional and post-transcriptional regulatory profiles between significantly differential phenotype of milk protein concentration provides new insights into the mechanism of milk protein synthesis, which should reveal the regulatory mechanisms of milk secretion.

scholarly journals Integrated Small RNA Sequencing, Transcriptome, and GWAS Data Reveal miRNA Regulation in Response to Milk Protein Traits in Chinese Holstein Cattle

2021 ◽  
Author(s):  
Wentao Cai ◽  
Cong Li ◽  
Junya Li ◽  
Jiuzhou Song ◽  
Shengli Zhang
Keyword(s):  
Protein Synthesis ◽  
Rna Sequencing ◽  
Small Rna ◽  
Protein Concentration ◽  
Milk Protein ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Production Traits ◽  
Economic Traits ◽  
Chinese Holstein

Abstract BackgroundMilk protein is one of the most important economic traits in the milk industry. Our previous study has revealed some functional genes responsible for milk protein synthesis in mammals. Yet, the miRNA-mediated gene regulatory network for the synthesis of milk protein in mammary is poorly understood. Results12 samples from Chinese Holstein Cows with three too high and three low phenotypic values for milk protein percentage in lactation and non-lactating were examined through deep small RNA sequencing. By bioinformatics analysis, we characterized 387 known and 212 novel miRNAs in the mammary gland. Differentially expressed analysis detected 28 miRNAs in lactation and 52 miRNAs in the non-lactating period with a highly significant correlation with milk protein concentration. Target prediction and correlation analysis identified some key miRNAs and their targets potentially involved in the synthesis of milk protein. Using genome-wide association signal (GWAS) enrichment analysis among five milk production traits, we found the differentially expressed targets were significantly related to milk protein traits.ConclusionsThis integrated study on the transcriptional and post-transcriptional regulatory profiles between significantly differential phenotype of milk protein concentration provides new insights into the mechanism of milk protein synthesis, which should reveal the regulatory mechanisms of milk secretion.

scholarly journals Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 964
Author(s):  
Sarka Benesova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
High Sensitivity ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Liquid Biopsies ◽  
Comprehensive Overview ◽  
Rna Molecules ◽  
Novel Mirna ◽  
The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

scholarly journals Small RNA sequencing evaluation of renal microRNA biomarkers in dogs with X-linked hereditary nephropathy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Candice P. Chu ◽  
Shiguang Liu ◽  
Wenping Song ◽  
Ethan Y. Xu ◽  
Mary B. Nabity
Keyword(s):  
Small Rna ◽  
Target Genes ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Ckd Progression ◽  
Critical Pathways ◽  
Qrt Pcr ◽  
Hereditary Nephropathy ◽  
Differentially Expressed Mirnas

AbstractDogs with X-linked hereditary nephropathy (XLHN) are an animal model for Alport syndrome in humans and progressive chronic kidney disease (CKD). Using mRNA sequencing (mRNA-seq), we have characterized the gene expression profile affecting the progression of XLHN; however, the microRNA (miRNA, miR) expression remains unknown. With small RNA-seq and quantitative RT-PCR (qRT-PCR), we used 3 small RNA-seq analysis tools (QIAGEN OmicSoft Studio, miRDeep2, and CPSS 2.0) to profile differentially expressed renal miRNAs, top-ranked miRNA target genes, and enriched biological processes and pathways in CKD progression. Twenty-three kidney biopsies were collected from 5 dogs with XLHN and 4 age-matched, unaffected littermates at 3 clinical time points (T1: onset of proteinuria, T2: onset of azotemia, and T3: advanced azotemia). We identified up to 23 differentially expressed miRNAs at each clinical time point. Five miRNAs (miR-21, miR-146b, miR-802, miR-142, miR-147) were consistently upregulated in affected dogs. We identified miR-186 and miR-26b as effective reference miRNAs for qRT-PCR. This study applied small RNA-seq to identify differentially expressed miRNAs that might regulate critical pathways contributing to CKD progression in dogs with XLHN.

scholarly journals Small RNA Sequencing Uncovers New miRNAs and moRNAs Differentially Expressed in Normal and Primary Myelofibrosis CD34+ Cells

PLoS ONE ◽  
2015 ◽  
Vol 10 (10) ◽  
pp. e0140445 ◽  
Author(s):  
Paola Guglielmelli ◽  
Andrea Bisognin ◽  
Claudia Saccoman ◽  
Carmela Mannarelli ◽  
Alessandro Coppe ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Primary Myelofibrosis ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Cd34 Cells

scholarly journals Small RNA Sequencing Reveals Differentially Expressed miRNAs in Necrotizing Enterocolitis in Rats

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Ren-qiang Yu ◽  
Min Wang ◽  
Shan-yu Jiang ◽  
Ying-hui Zhang ◽  
Xiao-yu Zhou ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Rna Sequencing ◽  
Necrotizing Enterocolitis ◽  
Small Rna ◽  
Mirna Expression ◽  
Expression Patterns ◽  
Wnt Signaling Pathway ◽  
Differentially Expressed ◽  
Small Rna Sequencing

Necrotizing enterocolitis (NEC) is the leading cause of death due to gastrointestinal disease in preterm infants. The role of miRNAs in NEC is still unknown. The objective of this study was to identify differentially expressed (DE) miRNAs in rats with NEC and analyze their possible roles. In this study, a NEC rat model was established using Sprague-Dawley rat pups. Small RNA sequencing was used to analyze the miRNA expression profiles in the NEC and control rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to identify target mRNAs for the DE miRNAs and to explore their potential roles. The DE miRNAs were verified by real-time quantitative PCR (RT-qPCR). The status of intestinal injury and the elevated levels of inflammatory cytokines in the NEC group confirmed that the NEC model was successfully established. The 16 miRNAs were found to be differentially expressed between the NEC group and the control group of rats. Bioinformatics analysis indicated that the parental genes of the DE miRNAs were predominantly implicated in the phosphorylation, cell migration, and protein phosphorylation processes. Moreover, the DE miRNAs were mainly found to be involved in the pathways of axon guidance, endocytosis, and focal adhesion, as well as in the Wnt signaling pathway, which is related to colitis. The expression patterns of the candidate miRNAs (rno-miR-27a-5p and rno-miR-187-3p), as assessed by RT-qPCR, were in accordance with the expression patterns obtained by miRNA-sequencing. The miRNA/mRNA/pathway network revealed that rno-miR-27a-5p and rno-miR-187-3p might be involved in NEC via the Wnt signaling pathway. We found an altered miRNA expression pattern in rats with NEC. We hypothesize that rno-miR-27a-5p and rno-miR-187-3p might mediate the NEC pathophysiological processes via the Wnt signaling pathway.

Identification of Differentially Expressed and Spliced Genes with RNA Sequencing Analysis of CLL Specimens

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4582-4582
Author(s):  
Wei Liao ◽  
Gwen Jordaan ◽  
Artur Jaroszewicz ◽  
Matteo Pellegrini ◽  
Sanjai Sharma
Keyword(s):  
B Cells ◽  
Rna Sequencing ◽  
Differentially Expressed Genes ◽  
Fold Change ◽  
Differentially Expressed ◽  
Pcr Analysis ◽  
Sequencing Analysis ◽  
Rna Seq ◽  
Mrna Isoforms ◽  
Core Set

Abstract Abstract 4582 High throughput sequencing of cellular mRNA provides a comprehensive analysis of the transcriptome. Besides identifying differentially expressed genes in different cell types, it also provides information of mRNA isoforms and splicing alterations. We have analyzed two CLL specimens and a normal peripheral blood B cells mRNA by this approach and performed data analysis to identify differentially expressed and spliced genes. The result showed CLLs specimens express approximately 40% more transcripts compared to normal B cells. The FPKM data (fragment per kilobase of exon per million) revealed a higher transcript expression on chromosome 12 in CLL#1 indicating the presence of trisomy 12, which was confirmed by fluorescent in-situ hybridization assay. With a two-fold change in FPKM as a cutoff and a p value cutoff of 0.05 as compared to the normal B cell control, 415 genes and 174 genes in CLL#1 and 676 and 235 genes in CLL#2 were up and downregulated or differentially expressed. In these two CLL specimens, 45% to 75% of differentially expressed genes are common to both the CLL specimens indicating that genetically disparate CLL specimens have a high percentage of a core set of genes that are potentially important for CLL biology. Selected differentially expressed genes with increased expression (selectin P ligand, SELPLG, and adhesion molecule interacts with CXADR antigen 1, AMICA) and decreased (Fos, Jun, CD69 and Rhob) expression based on the FPKM from RNA-sequencing data were also analyzed in additional CLL specimens by real time PCR analysis. The expression data from RNA-seq closely matches the fold-change in expression as measured by RT-PCR analysis and confirms the validity of the RNA-seq analysis. Interestingly, Fos was identified as one of the most downregulated gene in CLL. Using the Cufflinks and Cuffdiff software, the splicing patterns of genes in CLL specimens and normal B cells were analyzed. Approximately, 1100 to 1250 genes in the two CLL specimens were significantly differentially spliced as compared to normal B cells. In this analysis as well, there is a core set of 800 common genes which are differentially spliced in the two CLL specimens. The RNA-sequencing analysis accurately identifies differentially expressed novel genes and splicing variations that will help us understand the biology of CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Integrated transcriptome and small RNA sequencing analyses reveal a drought stress response network in Sophora tonkinensis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ying Liang ◽  
Kunhua Wei ◽  
Fan Wei ◽  
Shuangshuang Qin ◽  
Chuanhua Deng ◽  
...  
Keyword(s):  
Drought Stress ◽  
Rna Sequencing ◽  
Small Rna ◽  
Stress Responses ◽  
Molecular Mechanisms ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Severe Drought ◽  
A Genome ◽  
Sophora Tonkinensis

Abstract Background Sophora tonkinensis Gagnep is a traditional Chinese medical plant that is mainly cultivated in southern China. Drought stress is one of the major abiotic stresses that negatively impacts S. tonkinensis growth. However, the molecular mechanisms governing the responses to drought stress in S. tonkinensis at the transcriptional and posttranscriptional levels are not well understood. Results To identify genes and miRNAs involved in drought stress responses in S. tonkinensis, both mRNA and small RNA sequencing was performed in root samples under control, mild drought, and severe drought conditions. mRNA sequencing revealed 66,476 unigenes, and the differentially expressed unigenes (DEGs) were associated with several key pathways, including phenylpropanoid biosynthesis, sugar metabolism, and quinolizidine alkaloid biosynthesis pathways. A total of 10 and 30 transcription factors (TFs) were identified among the DEGs under mild and severe drought stress, respectively. Moreover, small RNA sequencing revealed a total of 368 miRNAs, including 255 known miRNAs and 113 novel miRNAs. The differentially expressed miRNAs and their target genes were involved in the regulation of plant hormone signal transduction, the spliceosome, and ribosomes. Analysis of the regulatory network involved in the response to drought stress revealed 37 differentially expressed miRNA-mRNA pairs. Conclusion This is the first study to simultaneously profile the expression patterns of mRNAs and miRNAs on a genome-wide scale to elucidate the molecular mechanisms of the drought stress responses of S. tonkinensis. Our results suggest that S. tonkinensis implements diverse mechanisms to modulate its responses to drought stress.

scholarly journals Transcriptional Analysis Reveals Alteration of Lung Macrophages Following Mesenchymal Stem Cell Therapy in Neonatal Mice With Hyperoxia-Induced Injury

2020 ◽  
Author(s):  
Ali Al-Rubaie ◽  
Robert DeMatteo ◽  
Foula Sozo ◽  
Timothy Cole ◽  
Richard Harding ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Pathway Analysis ◽  
Small Rna ◽  
Differentially Expressed ◽  
Transcriptional Analysis ◽  
Small Rna Sequencing ◽  
Specific Gene ◽  
Lung Macrophages ◽  
Neonatal Mice ◽  
Neonatal Hyperoxia

Abstract Background Lung immaturity is one of the most serious consequences of growth restriction and premature birth. Preterm babies often require mechanical ventilation to survive, but exposure to high levels of oxygen can permanently damage the lungs and interrupts normal development. As lung macrophages play an important role in hyperoxic lung injury and repair, our objective was to use next generation sequencing (NGS) to identify changes in the macrophage transcriptome following neonatal hyperoxia, with and without treatment with human mesenchymal stem cells (hMSCs). We provide the first report of RNA-sequencing of lung macrophages following neonatal hyperoxia and hMSCs therapy. Methods Neonatal mice exposed to normoxia (21%O2) or hyperoxia (90% O2) from birth to postnatal day 4 were randomized to receive either hMSCs or vehicle via intratracheal delivery on postnatal day 4. Mouse lungs from normoxia and hyperoxia groups with and without hMSCs therapy were examined at day 14. RNA-sequencing was performed on flow-cytometric CD45+CD11b+CD11c+ sorted lung macrophages. Purified total RNA was used to construct barcoded multiplex-compatible sequencing libraries using: 1) Illumina Stranded mRNA Sample Preparation chemistry (for transcriptomics) and 2) Bio Scientific NEXTFlex Small RNA chemistry (for small RNA). Results Sorted CD45+CD11b+CD11c+ lung macrophages from hyperoxia-exposed neonatal mice showed differentially expressed macrophage genes and miRNA compared to mice exposed to normoxia or hyperoxia+hMSCs. The administration of hMSCs was found to differentially upregulate 421 genes and downregulate 651 genes in CD45+CD11b+CD11c+ lung macrophages from neonatal mice exposed to hyperoxia, compared to normoxia. Integrity pathway analysis (IPA) analysis of macrophage-specific gene pathways revealed the effectiveness of hMSCs in altering macrophage function towards an anti-inflammatory ‘M2’ phenotype. Small-RNA sequencing provided further evidence on the effects of hMSCs, where 1,098 small RNAs transcriptomes were expressed as either significantly up- or down-regulated in response to hMSCs therapy following hyperoxia-induced lung damage. Conclusions Pathway analysis of the predicted mRNA targets of differentially expressed genes provides insight into miRNAs that preferentially target several important pathways. These miRNAs will be functionally relevant for lung macrophages, and will provide a greater understanding of the interaction between macrophage genotype and the associated phenotypes in the setting of inflammation or tissue repair.

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