Project description:NDNs and LDNs were isolated by density gradient centrifugation of PBMCs from 8 patients affected by tubercolosis (TB). In detail, RNA was extracted from NDNs and LDNs by Maxwell® RSC miRNA Tissue kit (Promega, Madison, USA), following manufacturer’s instructions. RNA concentration and quality was measured using NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). Total RNA (600 ng) was reverse transcribed using the RT2 First Strand Kit (SABiosciences, Qiagen, UK). 84 human cytokines and chemokines were tested using the RT2 Profiler PCR Array Human Innate & Adaptive Immune Responses array (Qiagen). A semiquantitative real-time RT-PCR was performed using a real-time PCR detection system (QuantStudio 7 Flex Real-Time PCR System, Thermo Fisher Scientific) with a two-step thermal cycling: 95 °C for 10 min, followed by 40 cycles (95 °C for 15 sec, 60 °C for 1 min). Data were analysed using the RT² Profiler PCR Array Qiagen data analysis software, and ACTB and B2M as housekeeping genes.
Project description:Non-coding RNA profiling by microarray in an Affymetrix® miRNA Array, version 4.1 (Santa Clara, CA, USA), commercially available by Thermo Fisher Scientific (Waltham, MA) We aimed to profile the expression of the entire population of annotated small non-coding RNAs in the Temporal Cortex (TC) of Alzheimer diseased (AD) individuals compared to age-matched controls.
Project description:100 ng of total RNA was processed with the Human v3 miRNA Expression Assay using NanoString nCounter system (NanoString, USA). Expression of individual miRNA was measured by quantitative real-time PCR with TaqMan miRNA Expression kit (Applied Biosystems, Thermo Fisher Scientific, USA).
Project description:Affymetrix Human Gene 2.0 ST microarray (ThermoFisher Scientific, Waltham, MA, USA) was used to select differentially expressed genes.
Project description:We aimed to identify urinary exosomal miRNAs associated with PCa metastasis and develop a non-invasive risk-scoring model for PCa metastasis in this study. MiRNA profiles were examined using the Taqman low-density miRNA array (TLDA). Megaplex reverse transcription reactions and pre-amplification reactions were performed to increase the quantity of cDNA for miRNA expression analysis using the Megaplex PreAmp Primers Human Pool A and TaqMan PreAmp Master Mix (Thermo Fisher Scientific). MiRNA expression was evaluated via the TLDA panel A v2.0 (Thermo Fisher Scientific). Raw data were processed using the QuantStudio Real-Time PCR Software (Thermo Fisher Scientific) to determine a cycle threshold (Ct) value for each miRNA.
Project description:To investigate the molecular pathological mechanisms of irritable bowel syndrome with diarrhea (IBS-D) and elucidate the effects of acupuncture on IBS-D colonic mucosa protein abundance in rats, a label-free high-throughput liquid chromatography-tandem mass spectrometry (LC-MS)-based proteomics analysis was used to survey the global changes of colonic mucosa proteins between different groups. A Nano flow Ultimate 3000 HPLC (Dionex Corp, Sunnyvale, CA) coupled online to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used in this project.
Project description:LC-MS/MS data of digested human and rabbit serum samples. Experiments were conducted using Dionex UltiMate 3000 (Thermo Fisher Scientific, USA) LC system connected to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Peptide spectrum matching of MS/MS spectra of each file was searched against the UniProt reviewed Human protein database (TaxID: UP000005640) using the Sequest algorithm within Proteome Discoverer v 2.4 software (Thermo Fisher Scientific, San Jose, CA).
Project description:Age-matched K18-hACE2 transgenic mice were infected intranasally with different SARS-CoV-2 viruses, including (1) USA-WA1/2020 (WA) of lineage A, (2) New York-PV09158/2020 (NY) of lineage B.1.3, (3) USA/CA_CDC_5574/2020 (CA) of lineage B.1.1.7 and (4) hCoV-19/South Africa/KRISP-EC-K005321/2020 (SA) of lineage B.1.351. Mouse lungs were harvested on 3 days post infection (dpi). Total RNA was extracted using QIAgen RNeasy Plus Mini Kit and was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Resultant cDNA was used as the template along with RT2 SYBR Green ROX qPCR Mastermix (Qiagen) to perform RT² Profiler™ PCR Array Mouse Hypoxia Signaling Pathway (Qiagen) real-time PCR in Stratagene MX3000p qPCR system.
Project description:To explore the cellular pathways and the molecular functions affected by a novel biogenic amine, (3-HKA), we performed a label free quantitative (LFQ) proteomic analysis of mouse lymphatic endothelial cells (LEC), and primary dendritic cells, treated with IFNgammaplus or minus 3-HKA. Biological triplicates, of total cell lysates, were fractionated by one-dimensional gel electrophoresis (1DEF) and the “in gel” tryptic derived peptides analyzed by nano-LC-ESI-MS/MS on a Q Exactive HF quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). LFQ analysis highlighted that 3-HKA downregulated many of the inflammatory pathways, more notably JAK/STAT1 and NFkB, associated with IFNgamma activation.
