Microarray/gene expression analysis of RNA isolated with two different protocols from human blood sampled on PAXgene and Tempus.
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ABSTRACT: Blood from four healthy volunteers was sampled on both PAXgene and Tempus tubes after two hours of fasting and abstention from coffee, medication and exercise. The content of each tube was split in two aliquotes where one aliquot was isolated using the sampling systems' original protocol and the second aliquot using a modified common protocol. The aim of this study was to establish a common RNA isolation protocol for these two systems and investigate if it could reduce the differences in gene expression.
Project description:Blood from four healthy volunteers was sampled on both PAXgene and Tempus tubes after two hours of fasting and abstention from coffee, medication and exercise. The content of each tube was split in two aliquotes where one aliquot was isolated using the sampling systems' original protocol and the second aliquot using a modified common protocol. The aim of this study was to establish a common RNA isolation protocol for these two systems and investigate if it could reduce the differences in gene expression.
Project description:RNA isolation and purification steps greatly influence the results of gene expression profiling. There are two commercially available products for whole blood RNA collection, PAXgene and Tempus blood collection tubes, and each comes with their own RNA purification method. We examined the impact of RNA isolation methods on gene expression profiles. We demonstrated that peripheral blood RNA isoaltion can critically impact differential expression results, particularly in the clinical setting where fold-change differences are typically small and there is inherent variability within biological corhorts Experiment Overall Design: Comparison of 5 healthy control samples drawn directly in PAXgene or Tempus tubes vs. the same 5 healthy control samples drawn in Li Heparin tubes with no PHA stimulation.
Project description:This SuperSeries is composed of the following subset Series:; GSE12709: Differential gene expression profiles are dependent upon method of peripheral blood RNA isolation (PHA); GSE12710: Differential gene expression profiles are dependent upon method of peripheral blood RNA isolation (direct, heparin) Experiment Overall Design: Refer to individual Series
Project description:RNA isolation and purification steps greatly influence the results of gene expression profiling. There are two commercially available products for whole blood RNA collection, PAXgene and Tempus blood collection tubes, and each comes with their own RNA purification method. We examined the impact of RNA isolation methods on gene expression profiles. We demonstrated that peripheral blood RNA isoaltion can critically impact differential expression results, particularly in the clinical setting where fold-change differences are typically small and there is inherent variability within biological corhorts Experiment Overall Design: Whole blood from 7 healthy individuals was collected using Li Heparin tubes, and was incubated for 3 hours at room temperature with or without addition of 25 ug/mL of PHA. After incubation, samples were subsequently transferred to either Tempus or PAXgene tubes.
Project description:This study investigates how the baseline expression level of genes impact symptomatic outcome of flaviviral infection. Before given the YF17D vaccine, whole blood was extracted into tempus tubes from vaccinated subjects. Thereafter, the subjects were assessed if they experienced adverse events (or symptoms). 27 subjects (n=19 symptomatic, n=8 asymptomatic) were processed according to manufacturer's protocol. Transcript expression was then evaluated by Affymetrix Human GeneChip 2.0 ST array, performed at Duke-NUS Genome Biology Core Facility. Data analysis and processing was performed using Partek software and enriched pathways determined by Reactome database from Enrichr.
Project description:The objective of this study was to identify specific gene expression profiles able to predict the response of rheumatoid arthritis patients treated with methotrexate /abatacept (Aba). Thirty six RA patients were received Abatacept. The drug efficacy was evaluated with the DAS28 score after 6 months of treatment according to the EULAR response criteria. Among 36 Aba RA patients, 17 were responders and 19 were classified as no-responders. A blood sample was carried out in patients just before the first injection of treatment in PaxGene tubes. Two color experiments : patient(Cy5)/Control pool (Cy3).
Project description:The autosomal recessive immuno-osseus dysplasia spondyloenchondrodysplasia (SPENCD) is characterised by the variable combination of metaphyseal and vertebral bone lesions, immune dysfunction with features of both autoimmunity and immunodeficiency, and neurological involvement including developmental delay and spasticity with intracranial calcification and leukodystrophy. This transcription profiling study of blood compared four patients to two control subjects. A deficiency of ACP5 encoding tartrate resistant acid phosphatase (TRAP) was found to cause this skeletal dysplasia demonstrating a type I interferon signature with autoimmunity.
Project description:Transcriptional profiling of peripheral blood mononuclear cells (PBMCs) from 30 subjects collected at day 0, day 1 and day 3 post yellow fever (YF) vaccination. Each patient sample is performed in duplicate to reduce the chance of error. Briefly, the project investigates how cross-reactive JE antibodies that are generated from prior vaccination can influence YF live-attenuated vaccine (LAV). The groups are hence segregated based on the YF antibody titers after 1 month vaccination. Group 4 refers to the control group where individuals received only the YF LAV. Enhancing group refers to the treatment group (given JE followed by YF vaccine), where individuals had YF antibody titers higher than 99% confidence interval of the YF antibody titers of the control group. Non-enhancing group, on the other hand, refers to the treatment group where individuals had YF antibody titers within or lower 99% confidence interval of the control group. Our present study provides evidence that enhancing levels of cross-reactive antibodies from prior Japanese encephalitis (JE) vaccination can improve subsequent YF immunogenicity. These microarray results indicate the genes and gene signatures that are associated with the differences in YF immunogenicity.
Project description:Assessment of transcriptome stability in Paxgene collected whole blood and representation of hematopoietic lineages of whole blood transcriptome using single primer isothermal amplification probes without globin reduction protocols. As well as the stability assessment of Paxgene collected samples we investigated whether expression profiling of whole blood RNA (without globin reduction procedures) using the NuGEN single-primer isothermal amplification methods (which generate single stranded cDNA) allows faithful representation of individual lineages. ie. are we losing a lot of information by looking at whole blood rather than PBMCs or individual lineages. These 7 cell lineages were not stored in Paxgene tubes and are only for the purpose of querying the data generated from the Paxgene/sscDNA dataset (12 samples) in order to determine the representation of individual lineage transcriptomes in that dataset.
Project description:In almost every countries the proportion of people over 60 years is growing faster that any other age group. Increased life expectancy is leading to the characterization of specific aspects of aging for the various physiological systems. The study of healthy aging is important to design strategies capable to maximize the health and to prevent chronic diseases in older people. Immunosenscence reflects the age-related changes of the immune system and the reduced capacity of elderly people to cope with new infections. To elucidate changes in gene expression related to systemic aging and immunosenescence in an unbiased manner we performed comparative microarray analysis on whole blood cell from healthy middle-aged versus elderly men, and correlated results with functional measurements of aerobic capacity. Blood cells from elderly subjects showed age-related changes in the expression of several markers of immunosenescence, inflammation and oxidative stress, and showed impairments in metabolic and biosynthetic capacities. Whole blood samples from twenty healthy men between the ages of 45-55 (n=11) and 65-75 (n=9) years old were analyzed for this study. For each subject blood was sampled in two different occasions, which were separated by at least 7 days. To preseve RNA quality and integrity 3 ml of blood have been collected intoTEMPUS Blood RNA tubes (ABI, Foster City, CA, USA). Total RNA has been depleted of globin mRNA using the GLOBINclearâ¢-Human kit (Ambion, Austin, TX). To reduce variations due to confounding events, such as spontaneous up- and down-regulation of genes, the two samples of clean RNA from each subject were pooled into one single sample (1ug+1ug RNA).