ABSTRACT: We compared 4 pools of plasma in the same iTRAQ proteomics experiment. The pool 1 contained plasma from twelve patients with strict BOS and with plasma sample available at onset of the clinical symptoms (named BOS), pool 2 contained plasma from sixteen patients with pulmonary infections (infection), pool 3 contained plasma from fifteen patients with chronic GVHD without pulmonary involvement (CGVHD no lung), and pool 4 contained plasma from fifteen patients after transplant with no chronic complications (at similar time point as BOS samples) (no complications).
Project description:We compared 4 pools of plasma in the same iTRAQ proteomics experiment. The pool 1 contained plasma from twelve patients with strict BOS and with plasma sample available at onset of the clinical symptoms (named BOS), pool 2 contained plasma from sixteen patients with pulmonary infections (infection), pool 3 contained plasma from fifteen patients with chronic GVHD without pulmonary involvement (CGVHD no lung), and pool 4 contained plasma from fifteen patients after transplant with no chronic complications (at similar time point as BOS samples) (no complications).
Project description:We compared 4 pools of plasma in the same iTRAQ proteomic experiment. The pool 1 contained plasma from twelve patients with strict BOS criteria and with plasma sample available at onset of the clinical symptoms (named BOS), pool 2 contained plasma from sixteen patients with pulmonary infections (infection), pool 3 contained plasma from fifteen patients with chronic GVHD without pulmonary involvement (CGVHD no lung), and pool 4 contained plasma from fifteen patients after transplant with no chronic complications (at similar time point as BOS samples) (no complications). Each pool contained 25 μl of plasma.
The four pooled plasmas were then individually immunodepleted of the twenty common hyper-abundant proteins with a ProteoPrep®20 plasma immunodepletion kit (Sigma-Aldrich) according to manufacturer?s procedure. The cysteine residues were alkylated and all samples were trypsinized. Each pool was labeled with a different tag allowing for differential quantification. The samples were labeled in the following order: 1) BOS with 114, 2) Infection with 115, 3) cGVHD with 116, and 4) No complication with 117. The four pooled plasma samples were dissolved in buffer A (7mM potassium phosphate, 30% acetonitrile, pH 2.65) and combined right before fractionation with a SCX column (Zorbax 300-SCX 5µm, 2.1 x 150 mm, Agilent). The fractions were collected every minute at 200 µL/min flow rate from 1% solvent B (7mM potassium phosphate, 500 mM KCl, 30% acetonitrile, pH 2.65) to 60% over 40 minutes (1% B for 7 minutes, 6 to 15% B for 18 minutes, 15 to 34% B for 10 minutes, and 34 to 60% B for 5 minutes) as well as during column washing at 98% solvent B for 10 minutes. These fractions were consolidated into 11 fractions using the UV trace to distribute the peptide quantities to be similar.
LC-MS/MS analysis was performed with an Easy-nLC 1000 (Thermo Scientific) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific). The peptide sample was diluted in 30 µL of 2% acetonitrile and 0.1% formic acid in water and 8-4 µL was loaded onto the column in triplicates and separated using a two-mobile-phase system consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A 90 minute gradient from 7% to 35% B at a flow rate of 400 nL/min was used for chromatographic separations. The mass spectrometer was operated in a data-dependent MS/MS mode over the m/z range of 400-1800. The mass resolution was set at 60,000. For each cycle, the 10 most abundant ions from the scan were selected for MS/MS analysis using 40% normalized HCD collision energy and analyzed with an orbitrap with the resolution set to 15000.
Project description:Chronic lung allograft dysfunction (CLAD) limits the long term survival after lung transplantation. CLAD is diagnosed late on the decline in lung function, when any immuno-intervention is ineffective. Identification of early forerunners of CLAD is therefore essential to prevent the progression of the disease before irreversible damages to the allograft. We used large-scale gene expression profiling of whole blood cells to identify early biomarkers of Bronchiolitis Obliterans Syndrome (BOS), the main form of CLAD. Microarray experiments performed from 80 patients (40 stable (STA) and 40 BOS) identify 47 genes differentially expressed between STA and BOS recipients. An independent set of patients (13 STA, 11 BOS) was then used for external validation by qPCR. POU Class 2 Associating Factor 1 (POU2AF1), T-cell leukemia/lymphoma protein 1A (TCL1A) and B-cell lymphocyte kinase (BLK) genes were identified and validated as predictive markers of BOS more than 6 months before diagnosis with AUCs of 0.83, 0.77 and 0.78 respectively. These genes allow stratifying upon CLAD risk (log-rank test p<0.01) and could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS. Overall design: This dataset represents gene expression profiling of periphal blood samples collected in PAXgene from 80 patients (40 stable (STA) and 40 BOS) using Agilent SurePrint G3 Human Gene Expression v3 8x60K Microarrays.
Project description:Brain injury resulting from hemorrhagic stroke is clinically challenging to manage and results in high rates of morbidity and mortality. The pathophysiology of brain damage resulting from aneurysmal subarachnoid hemorrhage (aSAH) is largely unknown, and methods to treat and monitor patients are variable with no meaningful correlations to patient outcome. Prediction of patient risk for serious neurological complications is currently a significant clinical obstacle. An extracellular RNA (exRNA) biomarker to predict onset and severity of brain damage would improve patient outcomes. We sequenced plasma and CSF samples from adult patients with SAH. Samples were collected from post bleed day 1 to day 7. Total exRNA was isolated from each sample. In addition, we prepared a subset of 140 CSF samples, isolating the RNA contained within extracellular vesicles and vesicle-depleted biofluid. Overall design: 523 total samples from 30 donors were analyzed. All patients suffered subarachnoid hemorrhages (no controls). Samples are either plasma or CSF. -------------------------------- Submitter states "We are currently working on submitting the data for these protected datasets to dbGaP, but wanted to submit the processed data to GEO in the mean time."
Project description:To determine the expression of circulating miRNAs in AIS patients with thrombolysis and without thrombolysis. Grant ID: 81100882 Grant title: The relation between transcription of miR-497 and neurons apoptosis after ischemic stroke Funding source: Chinese National Natural Science Foundation Grantee First Name: Zhen Grantee Last Name:Deng Overall design: Fifteen AIS patients with thrombolysis treatment and fifteen AIS patients (admission after 8h of onset) without thrombolysis were collected blood sample on the onset of 24h. Fifteen age-match healthy person were as control group. We used miRNA microarrays to detect plasma miRNA expression profiles and real-time PCR verified miRNA expression. Totally, there 3 sample in each group.
Project description:This study aimed to investigate the expression of microRNAs (miRNAs) in the plasma from polymyositis (PM) and dermatomyositis (DM) patients, which fluctuated by treatment. More differentially expressed miRNAs were found in plasma of DM patients compared to PM patients before and after treatment, and their profiles were different. Overall design: Total RNA was isolated from plasma of PM/DM patients before and after treatment with prednisolone, or, in case of prednisolone resistance or complications, with the combination of calcineurin inhibitors (cyclosporine or tacrolims) and/or pulse intravenous cyclophosphamide.
Project description:RNA was isolated from 200μl plasma samples and cDNA was synthesized. Real-time RT-PCR analysis was performed to evaluate miRNA expression in the plasma pool from 17 RA patients with RA-ILD or the plasma pool from 17 RA patients without ILD using Human miRNome microRNA PCR Panel I+II (Exiqon). Overall design: qPCR miRNA expression profiling. Plasma samples from 17 donors were used in control and test.
Project description:Long noncoding RNA profile in the plasma of human with hypertension and healthy controls from South China. Overall design: In the study presented here, we identified the genome-wide expression level of lncRNAs for one hypertension sample and one healthy control sample. Each sample (total RNA) was pooled from the total RNA of fifteen age and gender matched subjects (hypertension patients or healthy controls).
Project description:We performed whole-genome sequencing of sixteen individuals drawn from a 13-generation pedigree. Fifteen of these individuals comprise five parent-offspring trios. We generated 10-15X effective sequence coverage of the genome for each of these sixteen individuals using Illumina HiSeq paired-end reads. Using these sequence data, we have performed genetic variant detection for single nucleotide variants, indels, and copy number variants.
Project description:The aim of this study is to determine the molecular mechanism by which indoxyl sulfate acts on endothelial cells. Endothelial cells (HUVECs) were incubated with medium containing indoxyl sulfate at concentration found in chronic kidney disease patients (1mM). Since indoxyl sulfate is a potassium salt, control medium contains KCl at 1mM. Cells were incubated during four hours in presence of indoxyl sulfate or KCl. Seven biological replicates were obtained for these two conditions. To understand how uremic solutes participate in endothelial dysfunction and cardiovascular complications of chronic kidney disease patients