Lethal Nipah Virus Infection Induces Rapid Overexpression of CXCL10

Mathieu, Cyrille; Guillaume, Vanessa; Sabine, Amélie; Ong, Kien Chai; Wong, Kum Thong; Legras-Lachuer, Catherine; Horvat, Branka

Amplified and biotin-labeled RNAs were obtained from 2 mg of total RNA, by a round of in vitro transcription (dIVT) using the Message Amp a RNA kit version II (Ambion), following the manufacturer's protocol. Different quantities of positive synthetic mRNA controls (spikes, corresponding to 6 bacterial RNAs, used to control sensitivity, quality of hybridization and data normalization) were added to all samples, during the first step of reverse transcription of total RNAs. Hybridization was performed on Codelink Uniset Human Whole Genome bioarrays (http://www. codelink bioarrays.com). 10 mg of biotin-labeled RNA were fragmented using 5 ml fragmentation buffer in a final volume of 20 ml, then mixed with 240 ml Amersham hybridization solution and injected onto Codelink Uniset Human Whole Genome bioarrays, containing approximately 55.000 30-mer probes based on the NCBI/Unigene RefSeq database that permit the expression analysis of 57,347 transcripts and ESTs (GE Healthcare Europe GmbH). Arrays were hybridized overnight at 37uC, then washed in stringent TNT buffer at 46uC for 1 h before performing a streptavidin-cy5 detection step. Each array was incubated for 30 min in 3,4 ml streptavidin-cy5 solution, washed four times in 240 ml TNT buffer, rinsed twice in 240 ml water containing 0.2 M Triton X-100, and then, dried by centrifugation at 650 g. Arrays were then scanned at 635 nm using an Axon Genepix 4000B Scanner (Axon).