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: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: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 screen the differentially expressed lncRNAs, we performed lncRNA profiling using ArrayStar Human LncRNA Microarray in 24 new-onset systemic lupus erythematosus (SLE) patients and 12 age- and sex-matched healthy controls (HCs). For the lncRNA microarray screening, total RNA from plasma was isolated from 12 SLE without nephritis, 12 lupus nephritis (LN) and 12 HCs. Four RNA samples were mixed togther as a pool of sample for microarray analysis. Accordingly, there were each three pooled RNA samples from 12 SLE without nephritis, 12 LN and 12 HCs for microarray analysis. Hierarchical clustering showed the plasma levels of lncRNAs and mRNAs differed significantly between 24 new-onset SLE patients and 12 control subjects. With a fold change ≥ 2 and P ≤ 0.05, we identified 1315 significantly differentially expressed lncRNAs (743 lncRNAs up-regulated and 572 lncRNAs down-regulated) and 1363 differentially expressed mRNAs (745 mRNAs up-regulated and 618 mRNAs down-regulated) in plasma of SLE patients compared with control samples. Overall design: The human LncRNA microarray analysis of the 9 plasma samples from systemic lupus erythematosus patients and healthy controls
Project description:Arsenic (As) is a toxic environmental contaminant and potential human carcinogen. Chronic intake of arsenic-contaminated water leads to arsenicosis that is a major public health problem in many parts of the world, including India and Bangladesh. Thus, the early detection of arsenic toxicity will greatly benefit patients. However, the detection of arsenicosis needs to be done early before onset of severe symptoms in which case the tools used for detection have to be both sensitive and reliable. In this context, the present study investigated plasma proteome changes in arsenic-exposed Labeo rohita, with the aim of identifying biomarkers for arsenicosis. Changes in the plasma proteome were investigated using gel-based proteomics technology. Using quantitative image analysis of the 2D protein profiles, unique protein spots were identified from the plasma proteome by MALDI-TOF-TOF-MS. Unique proteins identified included Apolipoprotein-A1 (Apo-A1), α-2 macroglobulin-like protein (A2ML), and transferrin. Highly up-regulated protein spots identified in plasma from arsenic-exposed fish were liver-specific, including Apo-A1, and A2ML consistent with liver damage. It is proposed that a combination of these proteins could serve as useful biomarkers of hepatotoxicity and chronic liver disease due to arsenic exposure.
Project description:The early-onset mechanisms of idiopathic chronic pancreatitis, after the exclusion of known genetic risk factors, are rather unclear. Therefore, we attempt to pursue other potential pathology in our study and screened the differentially microRNAs from plasma samples of early-onset and late-onset ICP by microarray.
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:Rationale: Circular RNAs are pervasively expressed in highly diverged eukaryotes. Circular RNAs are more stable in body fluids, however, the link between circular RNA and onset of atrial fibrillation has never been investigated. Objective: To identify plasma circular RNAs for diagnosing onset of atrial fibrillation after the cardiac surgery. Methods and Results: Plasma circular RNAs expression was investigated in participants underwent isolated off-pump coronary artery bypass grafting. First, we used microarray to screen 15 circular RNAs in 30 plasma samples for diagnosing new onset of atrial fibrillation. Quantitative polymerase chain reaction assay was then applied to evaluate the expression of selected circular RNAs. Hsa_circRNA_025016 was upregulated in patients with onset of atrial fibrillation, with a high diagnostic accuracy by area under the receiver operating characteristic curve. The satisfactory diagnostic performance of hsa_circRNA_025016 persisted in validation cohort. Kyoto Encyclopedia of Genes and Genomes biological pathway analysis indicated that hsa_circ_025016 could participate in melanogenesis, insulin secretion, and thyroid hormone signaling pathway. There was a positive correlation between hsa_circ_025016 and fast blood glucose in both cohorts. Conclusions: Hsa_circ_025016 is a novel biomarker of onset of atrial fibrillation after isolated off-pump coronary artery bypass grafting. Overall design: Plasma Circular RNAs before surgery were collected. Plasma of patients with postoperative atrial fibrilltion and Patients without postoperative atrial fibrilltion were detected the expression of circular RNAs.