asy Mini Kit for the BSL2 viruses. For H5N1 infections, total RNA was extracted with Trizol LS. mRNAs were labeled with 33P for the reverse transcription using the Superscript III RT, dCTP and an oligodT25. Generated cDNAs were CAL-101 PI3K inhibitor hybridized on home made Nylon microarrays containing 9216 spotted IMAGE CAL-101 PI3K inhibitor human cDNA clones, representing 8682 genes and 434 control clones. Further details on the HuSG9k microarray are available on the TAGC website. All membranes used in this study belonged to the same batch. After hybridization and exposure on Micro Imager, arrays were scanned in a Fuji BAS 5000 machine and hybridization signals quantified using the BZ Scan Software.
Primary data, in accordance with the proposed MIAME standards, are accessible through GEO Series accession number GSE22319.
3 Data normalization and analysis Data files were loaded and analyzed CUDC-101 with R and Bioconductor , using the NylonArray library developed by the TAGC to support BZScan2 files. Raw data CUDC-101 were normalized by quantile normalization. Supervised analysis between groups Infected and Mock samples was conducted using the Significance Analysis of Microarray algorithm, using the siggenes library . All statistical analyses involved corrections for multiple comparisons . Agglomerative hierarchical clustering was performed by the pairwise average linkage method using the Pearson correlation distance.
4 Quantitative real time RT PCR validation To validate the microarray results with real time RT PCR assay, another set of A549 cells were infected with influenza viruses at a moi of 1 and total cell RNA was extracted at 24 hpi with Trizol LS.
Five hundred ng of total RNA were reverse transcribed using oligo18 and RevertAid M MuLV according to the manufacturer,s instructions. One mL of cDNA was then amplified and analyzed in the 7500 Real Time PCR System using the Platinum SYBR Green qPCR SuperMix UDG kit according to the manufacturer,s instructions. Six genes were chosen according to their level of expression and the availability of primers for the quantitative PCR. Glyceraldehyde 3 phosphate dehydrogenase mRNA was used as an internal control.
The reaction mix contained a total volume of 20 mL and the thermal cycling consisted of UDG incubation at 50uC for 2 min, 40 cycles of 95uC for 15 s and 60uC for 33 s for amplification. All data were normalized to the internal standard GAPDH mRNA.
For each single well amplification reaction, a threshold cycle was observed in the exponential phase of amplification. Relative changes in gene expression were determined using the 2DDCt method as previously described and reported as the n fold difference relative to a control cDNA prepared in parallel with the experimental cDNAs. Statistical significance was calculated using Welch,s two sample t test between mock and infected samples using R software. 5 In silico experiment: query the Connectivity Map with the infection signature To select potential antivirals, an unbiased in silico search for molecules that reverse the infection signature identified in the present study was performed using the publicly available Connectivity Map database . The Connectivity Map is a collection of genome wide transcriptional data from cultured human cells treated with different kinds of molecules. The 20 most differential