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Genome-wide profiling of gene expression under electro-acupuncture treatment on MI heart in rats

ABSTRACT: Purpose: Acupuncture exerts cardioprotective effects on several types of cardiac injuries, especially myocardial ischemia (MI). In order to elucidate the potential mechanisms, RNA-seq by next generation sequencing was used to identify the rat genome-wide alterations after MI and EA treatment in this study Methods: Adult male Sprague Dawley rats (250-300g) were divided into three groups: Control, MI and electro-acupuncture (EA) groups, myocardium mRNA profiles of rats in each group were generated by deep sequencing,using Illumina Hiseq 2000. 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. Results: Using an optimized data analysis workflow, we mapped about 25 million sequence reads per sample to the mouse genome (UCSC RN4) and identified 121,233, 178,242, 161,880 transcripts in the Control, MI and EA rats with TopHat and Cufflinks workflow respectively. Approximately 32% and 22% of the transcripts showed differential expression between the Control and MI, MI and EA respectively with a fold change ≥2.0. Conclusions: Our results for the first time generated genome-wide gene expression profiles both in the rat MI model and in acupuncture treatment by high throughput sequencing. The optimized data analysis workflows reported here should provide a framework for acupuncture investigations of expression profiles.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE54132 | GEO | 2016/03/21

SECONDARY ACCESSION(S): PRJNA235265

REPOSITORIES: GEO

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived WT and dKO round spermatids transcriptome profiling (RNA-seq) Methods: Adult WT and dKO round spermatids mRNA profiles mice were generated by deep sequencing, in dulplicate. 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: Using 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 (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and dKO round spermatids, 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. Conclusions: Our 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. Adult wild type (WT) and dKO mouse round spermatids were generated by deep sequencing, in dulplicate, using Illumina GAIIx.

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Genome-wide profiling of gene expression under electro-acupuncture treatment on MI heart in rats

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