Gene Expression
Nebulas
Gene Expression NebulasSummary: Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) is a syndrome of unknown etiology characterized by profound fatigue exacerbated by physical activity, also known as post-exertional malaise (PEM). Previously, we did not detect evidence of immune dysregulation or virus reactivation outside of PEM periods. Here we sought to determine whether cardiopulmonary exercise stress testing of ME/CFS patients could trigger such changes. ME/CFS patients (n=14) and matched sedentary controls (n=11) were subjected to cardiopulmonary exercise on 2 consecutive days and followed up to 7 days post-exercise, and longitudinal whole blood samples analyzed by RNA-seq. Although ME/CFS patients showed significant worsening of symptoms following exercise versus controls, with 8 of 14 ME/CFS patients showing oxygen consumption (V̇O2) on day 2, transcriptome analysis yielded only 6 differentially expressed gene (DEG) candidates when comparing ME/CFS patients to controls across all time points. None of the DEGs were related to immune signaling, and no DEGs were found in ME/CFS patients before and after exercise. Virome composition (P=0.746 by chi-square test) and number of viral reads (P = 0.098 by paired t-test) were not significantly associated with PEM. These observations do not support transcriptionally-mediated immune cell dysregulation or viral reactivation in ME/CFS patients during symptomatic PEM episodes.
Overall Design: RNAseq of whole blood samples from ME/CFS patients and controls following exercise.
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| Growth Protocol: | - |
| Treatment Protocol: | Cardiopulmonary exercise |
| Extract Protocol: | Whole blood (2.5 mL) from cases and controls was drawn into a PAXgene Blood RNA Tube (Qiagen, Valencia, CA) to stabilize RNA prior to extraction and stored at -20C. Total RNA was extracted using the PAXgene Blood RNA Kit (Qiagen, Valencia, CA) and all samples were lyophilized in RNAstable reagent (Biomatrica, San Diego, CA) for shipment at room temperature and long-term storage. |
| Library Construction Protocol: | The Ovation Human blood RNA-seq kit (Nugen, San Carlos, CA) was used to generate strand-specific RNA-seq libraries depleted for reads derived from rRNA (12S, 16S, 18S and 28S genes) and globin (HBA1, HBA2, HBB and HBD genes) according to the manufacturer^s protocol. Briefly, 100ng RNA extract, as measured by Qubit RNA high sensitivity kit (Thermo Fisher, South San Francisco), were DNased and cDNA was prepared from extracted total RNA by reverse transcription using a mixture of random and poly(T) primers. Successive steps of end-repair, adaptor ligation, strand selection via nucleotide analog-targeted degradation, insert-dependent adaptor cleavage for targeted depletion of rRNA and globin reads, PCR amplification, and bead-based purification were then used to construct cDNA libraries for RNA-seq analysis. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | paired |
| Library Strand: | Reverse; - |
| Platform: | Illumina |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Specific; Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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