final order: 76 cell type: Human bronchial epithelial cells (HBE) sample type: HBEs transfected with vRNA agent: vRNA moi: 5 time: 4 h
Treatment protocol
HBECs were stimulated with a 15 minute pulse of 1000U/ml IFNβ (PBL, Piscataway, NJ), 100ng/ml vRNA (purified directly from PR8 virus) with LTX transfection reagent (Invitrogen; Carlsbad, California), wild type H1N1 influenza (A/PR/8/34) or ΔNS1 virus (PR8 with a deleted NS1 gene, gift from Dr. Garcia-Sastre). Viruses were used at a multiplicity of infection (moi) of 5. Control samples were incubated with media or LTX under the same conditions. Cells were washed, supplemented with warm media and harvested at 11 timepoints (0, .25, .5, 1, 1.5, 2, 4, 6, 8, 12, and 18 hours post-treatment). HBECs were seeded in 6 well plates at a concentration of 250,000/well 18 hours prior to stimultaion. Cells were stimulated with a 15 minute pulse of IFNb, vRNA, infected with PR8 influenza or NS1 deleted influenza, or mock treated. In order to concentrate virus, red blood cells were used to bind virus for 30 minutes. Trypsin was then used to detach virus form the red blood cells. This was then spun at 1200 RPM for 5 minutes to separate RBCs from virus. The resulting supernatant was used to infect cells. As a control, we also made supernatant from RBCs that underwhent the same process but in the abcence of virus and used different ammount to treat cells for various times; these samples are included in this submission and are designated as pre (for pre trypsin) and post (for post trypsin). Analysis shows that there was no difference between these treatments and media alone.
Total RNA was extracted with QIAzol reagent following the miRNeasy kit’s procedure (Qiagen, Valencia, CA), and sample quality was tested on a 2100 Bioanalyzer (Agilent, Palo Alto, CA).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA (One-Cycle Target Labeling Assay, Affymetrix).
Hybridization protocol
Following fragmentation, 6 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human U133A HT Arrays. HT Arrays were washed and stained on the GeneChip Array Station
Scan protocol
GeneChips were scanned using the Affymetrix High Throughput Array Plate Scanner
Description
HBEs transfected with vRNA LTX+RNA4
Data processing
Data (in matrix) were normalized using RMA. Probes that were lower than 20 in all samples were removed and all values less than 20 were replaced by 20 for all remaining probes
