cell line: A549 cell type: alveolar adenocarcinoma cell line infection: A/Beijing/501/2009 (H1N1) time: 8 h post-infection
Treatment protocol
A549 cells were washed with phosphate-buffered saline (PBS) and subsequently infected with influenza A viruses at a MOI of 5 in a minimal volume of DMEM supplemented 0.5 mg/ml TPCK-treated trypsin (Sigma-Aldrich, USA) and 0.3% bovine serum albumin (Sangon, China) (infection medium). After one hour, cells were overlaid with fresh infection medium and incubated at 37°C for the indicated times.
Growth protocol
A549 cells were propagated in Dulbecco's Modified Eagle's Medium (DMEM; Life Technologies, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA) at 37°C in a 5% CO2 incubator.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted and purified using mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, US) following the manufacturer's instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US).
Label
Cy3
Label protocol
miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA, US) following the manufacturer's instructions, labeling section.
Hybridization protocol
Each slide was hybridized with 100ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA, US) in hybridization Oven (Agilent Technologies, Santa Clara, CA, US) at 55C, 20rpm for 20 hours according to the manufacturer's instructions, hybridization section. After hybridization, slides were washed in staining dishes (Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Agilent Technologies, Santa Clara, CA, US).
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Agilent Technologies, Santa Clara, CA, US) using the standard setting for 8x15k array slides.
Description
miRNA expression in lung epithelial cells after 8 hours of A/Beijing/501/2009 (H1N1) infection, replication 3.
Data processing
Fluorescence intensity was calculated using Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, US) with default settings and analysis performed using GeneSpring Software 11.0 (Agilent Technologies, Santa Clara, CA, US). Data normalization and transformation were all performed by the Shanghai Biochip Corporation.
