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Sequencing Facilitates Quantitative Analysis of lncSync-null and wild-type mouse heart cardiomyocytes

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE146032 | GEO | 2023/02/27

REPOSITORIES: GEO

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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.

Project description:Purpose: The current study tests the hypothesis that peroxisome proliferator-activated receptor Î² (PPARÎ²) has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. Methods: Wild-type (WT) and PPARÎ²-null mice (KO) male mice (3-5 month, C57BL/6) were kept in steel microisolator cages at 22Â°C with a 14-hr/10-hr light/dark cycle. Food and water were provided ad libitum throughout the entire feeding period. Standard PH was performed. All the animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under protocols approved by the University of California Davis Animal Care and Use Committee.Mouse liver RNA was prepared using TRIzol (Invitrogen, Carlsbad, CA). RNA concentration and integrity were determined by the Agilent 2100 Bioanalyzer using a RNA Nano Bioanalysis Chip. RNA-sequencing library preparation and sequencing was carried out by the Genome Sequencing Facility at University of Kansas Medical Center (Kansas City, KS). cDNA libraries were prepared with 2 Âµg of total RNA using the TruSeq RNA Sample Preparation Kit (Illumina). The libraries were clustered and sequenced on an Illumina HiSeq 2000 instrument with 100 bp paired end reads. Results: Differential gene expression profiling revealed the inhibition of expression in genes and pathways that are involved in metabolism and proliferation in regenerating KO livers. Specifically, PPARÎ² deficiency affected the activation of Akt and the expression of E2fs. Pathways that control glycolysis, FA synthesis as well as cell proliferation were de-regulated in regenerating PPARÎ² KO livers. Conclusions: The data suggest a role for PPARÎ² in regulating liver regeneration that is mediated at least in part through Akt and E2f-regulated pathways. mRNA profiles of 3-month old wild type (WT) and PPARbeta mice were generated by deep sequencing, one simple, using Illumina HiSeq 2000 instrument with 100 bp paired end reads

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Sequencing Facilitates Quantitative Analysis of lncSync-null and wild-type mouse heart cardiomyocytes

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