Monolayers of A549 cells (6 X 105 cells/ml) were infected with IAV at 5 X 102 TCID50, or 105 TCID50 IAV. The inoculum was removed, after one hour and the cells were washed twice with PBS and incubated with media containing 1 µg/ml TPEC trypsin for 30 minutes, 24 hours, or 48 hours at 37°C and 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the control and infected cells at 24 hours post-IAV infection using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and RNA was purified with the RNeasy kit (Qiagen, Valencia, CA). RNA purity and quantity was evaluated with Agilent chips (Agilent RNA 600 Nano kit).The purity and quantity of RNA were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip.
Label
streptavidin Alexa Fluor 647
Label protocol
Labeled cRNA was prepared using the MessageAmp II-Biotin enhanced kit (Ambion). 1.0 microgram of total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on columns. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA. The concentration of cRNA was determined by spectrophotometry. Streptavidin Alexa fluor 647 was incubated with the slides after hybridization to complete the labeling reaction. One sample was hybridized with each slide so no dye swaps were necessary.
Hybridization protocol
10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and once in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
Scan protocol
The slides were scanned in an Axon GenePix 4000B scanner using GenePix Pro 4.1 to align and acquire the microarray image. CodeLink Expression Analysis 4.1.0.4163 was used to apply the background correction.
Data processing
Data in the matrix table were prepared by global median normalization applied by CodeLink Expression Analysis 4.1.0.4163.
