Project description:The hedgehog pathway is crucial during airway epithelial cell differentiation. To assess the transcriptomic print of airway epithelial cells in absence of hedgehog pathway activation, we performed a comparative transcriptomic analysis on air-liquid interface cell cultures. Bronchial epithelial cells from 5 non-COPD subjects (ex- and current smokers) were isolated from bronchial brushes and cultured in air-liquid interface. The total RNA were extracted after 7 days of culture in air-liquid conditions in presence of an antibody targeting the sonic hedgehog ligand (AB5E1) or not (CTL). The libraries were prepared with NEBNext Ultra II directeional RNA Library Prep Kit and sequenced on Illumina.
Project description:Purpose: The goal of this study is to compare the effects of inhaled nicotine on aortic gene expression and the role of the alpha7 nicotinic acetylcholine receptor (alpha7-nAChR) in mediating the nicotine effects. Methods: WT and alpha7-nAChR knockout (KO) mice at 5-month of age were exposed to nicotine vapor or room air for a total of 12 weeks. Total RNA was extracted from thoracic aortas using RNeasy Plus mini kit followed by library preparation using SMART-Seq v4 Ultra Low Input RNA Kit. Illumina next generation sequencing was performed using the NextSeq 500 system with the high output flow cell (up to 800 M paired end reads). Results: A total of 179 differentially expressed genes (149 upregulated and 30 downregulated, adjusted P-value < 0.1) were found in thoracic aortas from WT mice exposed to nicotine vapor compared to those exposed to room air. In contrast, only 7 non-overlapping genes were found to be differentially expressed in alpha7-nAChR KO mice exposed to nicotine compared to the air controls. Conclusions: Our study suggests that nicotine-induced changes in vascular gene expression is largely mediated by the alpha7-nAChR.
Project description:Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. Here, the process of adaptation was investigated by determining changes in transcript profiles when aerobic steady-state cultures were depleted of air. Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow 1000 fermentation vessels (1.8 l capacity) with culture agitation speed constant at 300 rpm and the temperature maintained at 37 °C. Oxygen levels were monitored using galvanic oxygen electrodes while the pH was maintained at 7.2 ±0.2 by automatic titration with sterile KOH. Evans defined medium was used as the growth medium with glucose (15 mM) as the carbon source with the dilution rate being 0.2 h-1. Aerobic cultures were maintained by sparging the chemostat with air (0.4 l min-1). The switch to micro-aerobic conditions was achieved by switching off the air sparging the culture. After a period of 5, 10, 15 and 60 min exposure to air, cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA before total RNA purification using Qiagen’s Rneasy Midi kit as recommended by manufacturer’s instructions. Keywords: time-course, oxygen-depletion
Project description:Comparative small RNA seq analysis of WT and global p73KO Mouse Tracheal Epithelial Cell (MTECs) during the course of their differentiation (Air-Liquid Interface ALI D0, D4, D7, D14) aimed to determine the role of p73 in motile multiciliogenesis.
Project description:Serum miRNAs are considered useful as non-invasive biomarkers for various diseases, but the optimal method for extracting RNA from serum is currently unknown. In this study, several RNA extraction kits were used to determine which kit is the optimal method. RNA was extracted from the serum of 8-week-old C57BL/6NJcl male mice according to the protocol of each RNA extraction kit. The yield of extracted RNA samples was calculated and electrophoretic patterns were evaluated by Agilent bioanalyzer. Expression patterns of the extracted RNA samples were confirmed by Agilent mouse miRNA microarray. The results showed significant differences in RNA yields in the miRNeasy serum/plasma advanced kit, and mirVana™ PARIS™ RNA and Native Protein Purification Kit compared to almost all other samples. Furthermore, two peaks were identified in the miRNeasy serum/plasma advanced kit using small RNA kit of Agilent bioanalyzer, one at 20-40 nucleotides (nt) and the other around 40-100 nt whereas the other reagents had a single peak. In addition, a high correlation was observed between the two RNA extraction kits in microarray. These results suggest that the above two kits are suitable for miRNA extraction from mouse serum.