Project description:To further understanding of mature microRNA in plasma from healthy people, we have employed microRNA microarray as a discovery platform with the potential to identify the plasma microRNA levels of healthy people. Human peripheral blood was drawn from 4 healthy donors, and plasma was obtained by two-step centrifugation. Equal of total RNA from the plasma was detected by microRNA microarray including 866 human and 89 human viral miRNAs (Sanger miRBase, release 12.0). About 170 miRNAs could be detected by microarray in all 4 samples of plasma. Among them, 6 microRNAs (miR-451, miR-16, miR-133a, miR-1, miR-499 and miR-208a) were detected in the RNA from the same plasma by real-time PCR. Freshly peripheral bloods from 4 healthy donors were drawn into EDTA tubes, and were processed within 1 hour by two-step centrifugation to remove the blood cells and the cellular debris. Small RNA was prepared using the mirVana PARIS kit, and was quantified using a NanoDrop-1000 spectrophotometer. Equal of RNA from the plasma was detected by microRNA microarray to identify the microRNAs levels in plasma of healthy people.
Project description:We seek to discover small RNA biomarkers of autoimmune activity andor beta cell damage in type 1 diabetes. Pilot studies showed that heparinized plasma failed analyses, but that EDTA and citrated plasma did well, so 353 appropriate plasma samples average 11 per subject prospectively collected every 1 to 3 months from 32 high risk MAB or newly diabetic children and adolescents were collected, and the first 94 analyzed, for circulating small regulatory RNAs. 92 of 94 resulting cDNA libraries gave adequate numbers of miRNA mapped reads, but QC using spiked RNA internal standards showed abnormally high small RNA levels in 8 mildly hemolyzed plasma samples, leaving 84 of 94 with analyzable data. Equal numbers of EDTA and citrated plasma were analyzed successfully. Over the next period we will complete series on another 6 subjects, sequence the remaining 26070340 samples, and analyze the data for patterns of disease association with small RNA molecules in the prediabetic, perionset, and immediate post onset period. These patterns may identify biomarker small RNA predictive of autoimmune flares or beta cell loss, including predicting impending clinical onset.
Project description:Fasting blood samples were collected from 27 stroke patients in EDTA tubes mean 19 days post-stroke, plasma was extracted and frozen at -80C, then 200uL of plasma sent for qPCR.
Project description:Studies of miRNA profiling in the plasma of patients between ISR and non-ISR Venous blood was collected in EDTA in the ward or the cardiac catheterization laboratory before the angiography procedure and heparin administration. Plasma was harvested by centrifugation and stored at -80°C until assayed. Identical volumes of plasma from the 6 patients with ISR and 4 patients with non-ISR were pooled to reach a final volume of 1500µL for each patient. Total RNA was extracted using miRVana isolation kit, dephosphorylated and labeled using miRNA Complete Labeling kit. Scanning was achieved with the illumina iScan System. The result were acquired with the Genome Studio (GenomeStudioV2009.1).
Project description:High-throughput sequencing of the miRNAs present in plasma of COVID-19 patients at an early stage of the disease including non-SARS-CoV2 infected patients. This study allowed us to identify and functionally characterize human miRNAs associated with a worse evolution of the disease and a greater mortality. Samples were collected at hospital entry or within the first days after hospitalization and before treatment with immunotherapy for IL6 (e.g. Tocilizumab), interferon beta, corticoids and ribavirin, among others. Plasma samples were obtained from peripheral blood extracted in EDTA tubes after centrifugation. Total RNA, including small RNAs, was isolated from 400μl of plasma with the miRNeasy Serum Plasma Advanced kit (Qiagen). RNA quality and quantity were evaluated by the Bioanalyzer 2100 with Agilent RNA 6000 Nano Kit.
Project description:Peripheral blood was collected from 3 patients in two types of tubes, EDTA and Streck. After removing plasma, DNA was extracted from the remaining blood and subjected to array profiling
Project description:To further understanding of mature microRNA in plasma from healthy people, we have employed microRNA microarray as a discovery platform with the potential to identify the plasma microRNA levels of healthy people. Human peripheral blood was drawn from 4 healthy donors, and plasma was obtained by two-step centrifugation. Equal of total RNA from the plasma was detected by microRNA microarray including 866 human and 89 human viral miRNAs (Sanger miRBase, release 12.0). About 170 miRNAs could be detected by microarray in all 4 samples of plasma. Among them, 6 microRNAs (miR-451, miR-16, miR-133a, miR-1, miR-499 and miR-208a) were detected in the RNA from the same plasma by real-time PCR.
Project description:In this study we performed data-independet mass-spectrometry analysis of blood plasma collected from a cohort consisting of people living with HIV-1, people living with HIV-2, and HIV seronegative individuals. The data was used to infer signs of damage to a wide array of tissues and cell types.
Project description:Advancing Negative Ion Mode Proteomics. The main objective of the project is the exploration of the unconvetional negative ion mode for proteomics studies. In this work, we thoroughly studied the best chromatographic conditions for negative ion mode proteomics before testing different enzymatic digestion. The final goal is to establish the best working conditions in the negative polarity for negative ion mode. The method also refrains from any fragmentation events, which are unpredictable in negative ion mode.