Evidence for immune activation in pathogenesis of the HLA class II associated disease, podoconiosis
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Homo sapiens
SUBMITTER: Ben Towler
PROVIDER: E-MTAB-13860 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2024-11-21 | E-MTAB-14593 | biostudies-arrayexpress
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2024-02-28 | E-MTAB-13781 | biostudies-arrayexpress
Project description:To analyse gene expression pattern in different disease state of COVID-19 patients. Experimental workflow: 1) rRNA was removed by using RNase H method, 2) QAIseq FastSelect RNA Removal Kit was used to remove the Globin RNA, 3) The purified fragmented cDNA was combined with End Repair Mix, then add A-Tailing Mix, mix well by pipetting, incubation, 4) PCR amplification, 5) Library quality control and pooling cyclization, 6) The RNA library was sequenced by MGI2000 PE100 platform with 100bp paired-end reads. Analysis steps: 1) RNA-seq raw sequencing reads were filtered by SOAPnuke (Li et al., 2008) to remove reads with sequencing adapter, with low-quality base ratio (base quality < 5) > 20%, and with unknown base (’N’ base) ratio > 5%. 2) Reads aligned to rRNA by Bowtie2 (v2.2.5) (Langmead and Salzberg, 2012) were removed. 3) The clean reads were mapped to the reference genome using HISAT2 (Kim et al., 2015). Bowtie2 (v2.2.5) was applied to align the clean reads to the transcriptome. 4)Then the gene expression level (FPKM) was determined by RSEM (Li and Dewey, 2011). Genes with FPKM > 0.1 in at least one sample were retained.
2021-03-24 | E-MTAB-10298 | biostudies-arrayexpress
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Project description:Pneumonia remains the leading cause of death in children under five, but existing diagnostic methods frequently lead to either insufficient or excessive treatments. Our study aims to bridge this gap by identifying host transcriptomic biomarkers in the blood of children with confirmed viral or bacterial pneumonia. Using RNA sequencing, we identified and validated in an independent cohort a 5-transcript signature that accurately differentiates bacterial from viral pneumonia. This signature has considerable potential to improve diagnostic accuracy for pediatric pneumonia, minimizing delays in diagnosis and avoiding unnecessary treatments, with the possibility of significantly impacting clinical practice. a strand specific library preparation was completed using NEBNext® Ultra™ II mRNA kit (NEB) and NEB rRNA/globin depletion probes following manufacturer’s recommendations. Individual libraries were normalized using Qubit, pooled together and diluted. The sequencing was performed using a 150 paired-end configuration in a Novaseq6000 platform. Quality control of raw data was carried out using FastQC, alignment and read counting were performed using STAR, alignment filtering was done with SAMtools and read counting was carried out using FeatureCounts. RNAseq data was processed for batch correction using control samples and COMBAT-Seq package.
2024-12-08 | E-MTAB-14564 | biostudies-arrayexpress
Project description:In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. We compared technical and biological replicates having undergone globin depletion or not and found that globin depletion removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Peripheral whole blood transcriptome assessed by RNA-Seq on Illumina HiSeq 2000 in 6 healthy individuals and 6 pooled samples, either globin depleted or not.
2014-02-27 | E-GEOD-53655 | biostudies-arrayexpress
Project description:Microarray-based gene expression analysis of peripheral whole blood is a common strategy in the development of clinically relevant biomarker panels for a variety of human diseases. However, the results of such an analysis are often plagued by decreased sensitivity and reliability due to the effects of relatively high levels of globin mRNA in whole blood. Globin reduction assays have been shown to overcome such effects, but they require large amounts of total RNA and may induce distinct gene expression profiles. The Illumina whole-genome DASL (WG-DASL) assay can detect gene expression levels using partially degraded RNA samples and has the potential to detect rare transcripts present in highly heterogeneous whole blood samples without the need for globin reduction. We therefore assessed the utility of the WG-DASL assay in the analysis of peripheral whole blood gene expression profiles. We find that gene expression detection is significantly increased with the use of WG-DASL compared to the standard in vitro transcription-based direct hybridization (IVT), while globin-probe-negative WG-DASL did not exhibit significant improvements over globin-probe-positive WG-DASL. Globin reduction increases the detection sensitivity and reliability of both WG-DASL and IVT with little effect on raw intensity correlations: raw intensity correlations between total RNA and globin-reduced RNA were 0.970 for IVT and 0.981 for WG-DASL. Overall, the detection sensitivity of the WG-DASL assay is higher than the IVT-based direct hybridization assay, with or without globin reduction, and should be considered in conjunction with globin reduction methods for future blood-based gene expression studies. Peripheral whole blood samples were collected from eight human donors in PAXGene tubes. RNA was isolated after freezing and storage, and then prepared for gene expression analysis using the Illumina Human-Ref8 v3.0 BeadChip. Alpha and beta globin were reduced from a portion of the total RNA using the GLOBINclear assay (Ambion, Austin, TX, USA). Two methods of microarray target preparation were examined: Illumina IVT-based direct hybridization (IVT) and Illumina Whole-Genome DASL (WG-DASL). Two DASL Assay Oligo pools (DAP) were utilized for DASL target preparation: the DASL Assay Oligo Pool with globin probes (DAP +) and the DASL Asssay Oligo Pool without globin probes (DAP-).
2011-04-01 | E-GEOD-28064 | biostudies-arrayexpress
Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC There are 10 samples for each of PBMC, PAX and PAX globin-reduced, where 5 samples come from sickle-cell patients and 5 from healthy controls.
2009-06-20 | E-GEOD-16728 | biostudies-arrayexpress
Project description:The transcriptional responses of human hosts towards influenza viral pathogens are important for understanding virus-mediated immunopathology. Despite great advances gained through studies using model organisms, the complete temporal host transcriptional responses in a natural human system are poorly understood. In a human challenge study using live influenza (H3N2/Wisconsin) viruses, we conducted a clinically uninformed (unsupervised) factor analysis on gene expression profiles and established an ab initio molecular signature that strongly correlates to symptomatic clinical disease. This is followed by the identification of 42 biomarkers whose expression patterns best differentiate early from late phases of infection. In parallel, a clinically informed (supervised) analysis revealed over-stimulation of multiple viral sensing pathways in symptomatic hosts and linked their temporal trajectory with development of diverse clinical signs and symptoms. The resultant inflammatory cytokine profiles were shown to contribute to the pathogenesis because their significant increase preceded disease manifestation by 36 hours. In subclinical asymptomatic hosts, we discovered strong transcriptional regulation of genes involved in inflammasome activation, genes encoding virus interacting proteins, and evidence of active anti-oxidant and cell-mediated innate immune response. Taken together, our findings offer insights into influenza virus-induced pathogenesis and provide a valuable tool for disease monitoring and management in natural environments. During the challenge study, subjects had peripheral blood taken 24 hours prior to inoculation with virus (baseline), immediately prior to inoculation (pre-challenge) and at set intervals (8 hour intervals) following challenge. RNA was extracted at Expression Analysis (Durham, NC) from whole blood using the PAXgene™ 96 Blood RNA Kit (PreAnalytiX, Valencia, CA) employing the manufacturer's recommended protocol. While whole blood RNA is initially extracted, a secondary procedure (B-globin reduction) was then employed to remove the contribution of red blood cell (RBC) RNA to the total RNA. A set of four peptide nucleic acid (PNA) oligomers whose sequences are complementary to the 3’ portions of the alpha and beta hemoglobin RNA transcripts were added to reduce globin RNA transcription due to RBC. The inhibition of globin cDNA synthesis dramatically reduces the relative amount of anti-sense, biotin-labeled cRNA corresponding to the hemoglobin transcripts. Hybridization and microarray data collection was performed using the Human Genome U133A 2.0 Array (Affymetrix, Santa Clara, CA) and expression profiles were pre-processed using robust multi-array (RMA) method
2011-07-12 | E-GEOD-30550 | biostudies-arrayexpress