For RNA isolation, Calu-3 cells were seeded in 6-well plates (1 x 10^6 cells/well) two days prior to infection. Immediately preceding infection, monolayers were washed twice with DF12 supplemented with 0.3% bovine serum albumin (DF12-BSA), and inoculated with VN1203 (multiplicity of infection [MOI] of 1 plaque forming unit per cell) in DF12-BSA for 50 minutes at 37°C. Mock-infected controls were inoculated with DF12-BSA only. Following inoculation, monolayers were washed once with DF12-BSA and incubated in DF12-BSA containing 0.5 μg/ml of TPCK-treated trypsin (Worthington Biochemical Corporation, Lakewood, NJ) for the times indicated.
Growth protocol
Calu-3 cells, a human lung adenocarcinoma cell line, were kindly provided by Dr. Raymond Pickles (University of North Carolina, Chapel Hill, NC) and were maintained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 nutrient medium (DF12; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. All cells were grown at 37°C in an atmosphere of 5% CO2, with an antibiotic/antimycotic mixture (Invitrogen).
Extracted molecule
total RNA
Extraction protocol
At 0, 3, 7, 12, 18 and 24 hours post-infection (hpi), triplicate wells of mock-infected and VN1203-infected Calu-3 monolayers were washed with 5 ml cold phosphate-buffered saline (PBS) and lysed directly with 1 ml of TRIzol (Invitrogen), according to the manufacturer’s recommendation. The resulting lysates were stored at -80°C until further processing. All TRIzol lysates were processed simultaneously: they were phase-separated, and RNA was isolated from the aqueous phase (diluted 2 fold with RLT buffer) using Qiagen RNeasy Mini columns and the manufacturer’s recommended protocol (Qiagen Inc., Valencia, CA). RNA quality was assessed on an Agilent 2100 Bioanalyzer using the nanochip format, and only intact RNA was used for quantitative real-time PCR (qPCR) and microarray analyses.
Label
Cy3
Label protocol
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for all processing steps, including Cy3-cDNA probe preparation.
Hybridization protocol
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for all processing steps, including hybridization and array washing.
Scan protocol
Dry slides were scanned on an Agilent DNA microarray scanner (Model G2505B) using the XDR setting.
Description
251485048468_1_3 Mock host response 3H.
Data processing
Raw images were analyzed using the Agilent Feature Extraction software (version 9.5.3.1) and the GE1-v5_95_Feb07 extraction protocol. Data were normalized using RMA.
Submission date
Mar 24, 2011
Last update date
Sep 02, 2011
Contact name
Armand Bankhead III
Organization name
Oregon Health and Science University
Department
Depatartment of Medical Informatics and Clinical Epidemiology
