Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type, serpini1-/- and Tg(serpini1) zebrafish Transcriptomes
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE147351 | GEO | 2020/12/31
REPOSITORIES: GEO
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Project description:The development of predictive markers to determine the tipping point prior to the onset of osteoporosis is essential to mitigate further irrevocable deterioration. In order to determine the early warning signs before osteoporosis, we used the dynamic network biomarker (DNB) approach to analyze the time series gene expression data in the zebrafish osteoporosis model and revealed that cyclin-dependent kinase inhibitor 1A (cdkn1a) is one core member of DNB. We demonstrated that cdkn1a negatively regulates osteogenesis, evidenced by the loss-of-function and gain-of-function studies. On one hand, CRISPR/Cas9-mediated cdkn1a knockout in the zebrafish significantly altered skeletal development and increased bone mineralization. On the other hand, inducible cdkn1a expression significantly contributed to the osteoclast differentiation. We also provide mechanistic clues that cdkn1a participates in osteoclast differentiation by regulating its upstream signaling cascades. Our data provided new insights into the dynamic nature of osteoporosis, and identified cdkn1a as the early-warning signals of osteoporosis onset.
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Project description:Heart tissue mRNA profiles of 50 wild-type (WT) and 50 homozygous smyd4 mutant zebrafish embryos at 72-hpf created by CRISPR/Cas9 were generated by deep sequencing.
2018-01-25 | GSE109553 | GEO
Project description:Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type, serpini1-/- and Tg(serpini1) zebrafish Transcriptomes
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Project description:Our previous experiments showed the function of Aldh3a1 was related to pancreas and insulin secretion and knockout of Aldh3a1 led to destoryed pancreas. To understand how the impaired pancreas reflects at the transcriptome level, we performed full genome RNA-Seq between aldh3a1+/+ and aldh3a1-/- larvae at 48 hpf.An overview of RNA-Seq, including quality control, principal component analysis (PCA), volcano plots of regulated genes and Top 20 KEGG pathway analysis showed comparable properties between aldh3a1 mutants and wild type. We found the insulin signalling pathway was reduced significantly via gene set enrichment analysis (normalized enrichment score, -1.72; p=0.0006). Interestingly, endocrine pancreas but not exocrine pancreas development pathway was significantly down-regulated in aldh3a1 mutants . Taken together, these results further suggest Aldh3a1 as an important regulator in the development, morphology and insulin secretion function of primary pancreas. Furthermore, we offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within zebrafish larvae. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
2020-10-06 | GSE151770 | GEO
Project description:Next Generation Sequencing Facilitates Quantitative Analysis of Zebrafish Osteoporosis Model Transcriptomes
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Project description:Our previous experiments showed the function of Akr1a1a was related to insulin resistance. The knockout of Akr1a1a led to poor acrolein detoxification and accumulated acrolein inhibits insulin receptors (insra/insrb) expression. To understand how the loss of Akr1a1a and acrolein reflects at the transcriptome level, we performed full genome RNA-Seq between akr1a1a+/+, akr1a1a-/- and akr1a1a+/+ with acrolein treatment zebrafish larvae at 120 hpf. An overview of RNA-Seq, including quality control, principal component analysis (PCA), and volcano plots of regulated genes showed comparable properties between akr1a1a mutants, wild type and acrolein treated wild type zebrafish larvae. We found the insulin receptor signaling pathway was down-regulated significantly in akr1a1a mutants and acrolein treated wild type larvae as compared to wild type larvae via gene set enrichment analysis (normalized enrichment score, -1.888, p=0.028; -1.93, p=0.019). Intriguingly, downstream signaling pathways including MAPK, signal transduction by protein phosphorylation and transmembrane receptor protein tyrosine kinase signaling pathway were also significantly down-regulated in akr1a1a mutants and acrolein treated wild type larvae. Taken together, these results further suggest akr1a1a and acrolein as an important regulator in insulin receptor signaling transduction. Furthermore, we offer a comprehensive and more detailed evaluation of mRNA content within zebrafish larvae. We conclude that RNA-seq based transcriptome would clearly illustrate genetic network and clarify complex biological functions.
2021-09-05 | GSE168786 | GEO
Project description:PurposeNext-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods and to evaluate protocols for optimal high-throughput data analysis.MethodsRetinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl(-/-)) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows-Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT-PCR validation was performed using TaqMan and SYBR Green assays.ResultsUsing an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl(-/-) mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT-PCR. RNA-seq data had a linear relationship with qRT-PCR for more than four orders of magnitude and a goodness of fit (R(2)) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl(-/-) retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT-PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling.ConclusionsOur study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
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Project description:The goals of this study are to compare NGS in 3day post fertilized zebrafish embryo after knockout terfa (telomere repeats binding factor 2). In order to investigate the role of terfa in DDR, telomere protection and neuro-development. Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the zebrafish genome (build zv10) and identify 8840 NGS in 'WT vs terfa-/-' group and 4137 NGS in 'WT vs terfa+/-' group. Our results indicate that zebrafish terfa involved in 1. cell cycle dysregulation (e.g. senescence, p53, spliceosome, RNA transport, Ribosome, mRNA surveillance and cell cycles), including an increased expression of two p53-targeted genes, cdkn1a (p21) and gadd45; 2. inflammation (e.g. Salmonella and Herpes simplex infections, TOL-like receptor signaling and Cell Adhesion), including an increased expression of two pro-inflammatory genes, il6 and isg15. 3. neuronal dysfunction (e.g. Phototransduction, Calcium signaling and Neuroactive ligand-receptor interaction), including a signature of glial injury (gfap, ctsl, metrn, gcm2, clu and 5 members of the slc1a family (3, 4, 5, 7, 8)).
2022-03-09 | GSE173639 | GEO
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the brain of atrn wildtype and homozygous adult zebrafish transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Total RNA was isolated from single brain of the wildtype and atrn-/- zebrafish using TRIzol reagent (Invitrogen) respectively by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Bioinformatics analysis of the transcriptome sequencing found that the steroid synthesis pathway was enriched in results of the mutant brain. By analyzing the data of male and female zebrafish with atrn mutations, it was found that the expression levels of related genes in the steroid synthesis pathway were up-regulated, mainly including the gene encoding the key enzyme for cholesterol synthesis, cyp51 and the gene encoding the key enzyme for testosterone synthesis, hsd17b7.
2021-12-08 | GSE178506 | GEO
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the early stage of pEGFP-N1 injected zebrafish embros transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: we injected the plasmid pEGFP-N1 into zebrafish zygotes by microinjection, and then when the embryos to develop to 1 hour-post-fertilization (hpf), 6 hpf and 12 hpf, these groups was collected respectively by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Bioinformatics analysis of the transcriptome sequencing found that the expression levels of genes related to the apoptosis process (Fig.1A), endogenous immune response and tumor necrosis factor-mediated signaling pathways were abnormal up-regulation, mainly including isg15, foxo3b, phlda3, cdkn1a, zgc:136826, and si:dkey-204l11.1, compared with the non-injected group at 6hpf. These genes are found in somatic cells to deal with foreign plasmids invasion.
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