Statistical analysis of microarray data The cells were infected with either (A) the H1N1/2002 strain or (B) the H5N1/2004 strain, or (C)

mock-infected with PBS (no infection control). Cell samples were collected at 3, 6, 18 and 24 hours mTOR inhibitor review post-infection. Each miRNA array allowed us to interrogate 866 human miRNAs. The results were analyzed using Genespring GX 10.0.2 software (Agilent Technologies). Firstly, the 16 arrays were quantile normalized selleck chemicals llc together. Then, student’s paired t-test was applied to test if there was a significant difference between (A) the H1N1/2002-infected and (C) mock-infected, no infection control (matched for the time post-infection), (B) the H5N1/2004-infected and (C) mock-infected control, respectively. The resultant P-values were adjusted for multiple testing by using the Benjamini-Hochberg correction of the false-discovery rate [37]. MiRNAs with this adjusted P-value <= 0.05 were considered as differentially learn more expressed. Those miRNAs, that are more than or equal to 3.5-fold up or down regulated were subjected to a second analysis using real-time RT-PCR. MicroRNA profiling data resource The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [38] and are accessible through GEO Series accession number GSE44455. TaqMan Real Time RT-PCR (qRT-PCR) for quantification of miRNAs Total RNA was reverse

transcribed with looped miRNA-specific RT primers contained in the TaqMan MicroRNA assays ((Applied Biosystems, Foster City, CA). Briefly, single-stranded cDNA was synthesized from 10 ng total RNA in 15-μL reaction volume with TaqMan MicroRNA reverse transcription kit (Applied Biosystems), according to the manufacturer’s protocol. The reaction was incubated

at 16°C for 30 min followed by 30 min at 42°C and inactivation at 85°C for 5 min. Each cDNA was amplified Meloxicam with sequence-specific TaqMan microRNA assays (Applied Biosystems). PCR reactions were performed on an Applied Biosystems Step One sequence detection system in 10 μl volumes at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. All samples were tested in triplicate. The threshold cycle (Ct) values obtained with the SDS software (Applied Biosystems) were compared with the Ct obtained from 18S rRNA assay (Applied Biosystems) for the normalization of total RNA input. The fold-change was calculated based on Ct changes of mean medium Ct minus individual Ct of a miRNA. Each experiment was performed in triplicate. qRT-PCR for quantification of TGF-β2 mRNA level Total RNA extracted from cell cultures was reversely transcripted to cDNA using the poly(dT) primers and Superscript III reverse transcriptase (Invitrogen), and quantified by real-time PCR. The sense and antisense primers used in real-time PCR for measuring TGF-β2 were: (Forward: 5′-CCAAAGGGTACAATGCCAAC-3′; Reverse: 5′-TAAGCTCAGGACCCTGCTGT-3′).