Two-hundred and thirty microliters of sample was centrifuged at 17,000 g for 10 minutes. The supernatant was filtered through a 0.22 µm Ultrafree MC spin filter (Costar) at 2000 g for 2 minutes and DNase treated (5 U DNaseI (Invitrogen or New England Biolabs) for 30-1½ hours at room temperature (urine and CSF samples were considered free of genomic DNA and hence not DNAse treated). The viral nucleic acid (NA) was extracted using the PureLink Viral RNA/DNA kit (Invitrogen), without the addition of carrier RNA. The extracted viral NA was eluted with 30 µl DNase/RNase-free sterile water, and stored at -20 ºC or immediately used. Virus-positive supernatants suspended in RNA-extraction reagent were purified according to manufacturer (TRIzol, Life Technologies; TriFast, Peqlab; AVL buffer, Qiagen). The resulting RNA was further purified using QIAamp RNA viral Mini kit (Qiagen). The Extracted viral NA was eluted with 30µl DNase/RNase-free sterile water, and stored at -20 ºC or immediately used. To be able to amplify both DNA virus and RNA virus samples with the same protocol, we used the described Whole Transcriptome Amplification (WTA) method (Berthet et al. Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR. BMC Molecular Biology 2008, 9:77) by using the QuantiTect WTA kit (Qiagen) according to manufacturer’s protocol, except for the reverse transcription step that was replaced by the Superscript III Reverse Transcription kit (Invitrogen) combined with 5´-phosphorylated random hexamers (P-N6)(Eurofines MWG Operon). The WTA protocol includes 3 sequential reactions: firstly a reverse transcription reaction to generate cDNA, secondly a ligation of generated cDNA into larger transcriptomes and thirdly a Repli-g amplification of the generated transcriptomes. Neither of the two first reactions (cDNA synthesis and ligation) had any adverse effects when performed on a DNA-virus. Briefly, 11 µl of extracted viral NA was put into a 20 µl RT reaction and incubated at 42 ºC for 1 hour. The Superscript enzyme was inactivated at 95 ºC for 5 minutes, 10 µl of the cDNA reaction was added to 10 µl of ligation mix and incubated at 22 ºC for 2 hours. Thirty microliters of amplification mix containing Phi29 polymerase and random primers was then added and amplification performed at 30 ºC for 2-8 hours. The reaction was terminated by incubation at 95 ºC for 5 minutes. Amplified material was purified using QIAamp DNA mini kit (Qiagen) using a modified protocol (www.qiagen.com), and checked for purity and concentration using a NanoDrop spectrophotometer (Thermo Scientific) and agarose gel electrophoresis. The DNA was then stored at -80 ºC or immediately used for microarray analysis.
Label
Cy5
Label protocol
Labelling was performed in-house at SSI, Denmark, according to manufacturer’s protocol (Gene expression analysis, Roche NimbleGen). Briefly, 1 µg of heat-denatured WTA-amplified sample was labelled using nick translation with Cy3- or Cy5-labeled random nonamer primers and Klenow fragment (exo-) (NEB) at 37 ºC for 2 hours. Labelled DNA was iso-propanol precipitated and the pellet washed, dried, reconstituted and quantitated.
Hybridization protocol
Hybridization was performed in-house at SSI, Denmark, according to manufacturer’s protocol (Gene expression analysis, Roche NimbleGen). Briefly, 8 µg of labelled DNA was used for hybridization. Hybridization was performed in a NimbleGen Hybridization system using the NimbleGen Hybridization kit (Roche NimbleGen).
Scan protocol
Microarrays were scanned in-house at SSI, Denmark using a MS 200 Scanner (Roche, Nimblegen)
Description
Data used in Table 3
Data processing
Data was analyzed using NimbleScan software and custom software developed at Lawrence Livermore National Laboratory, CA, USA and described in 'Gardner et al. A Micobial Detection Array (MDA) for Viral and Bacterial Detection. BMC Genomics 2010 Nov 25;11:668'. Additional stringency criteria were applied to exclude bacterial sequences and viruses having fewer than 20% of probes detected out of those expected. Probes were classified as detected if the intensity exceeded a threshold equal to the 99th percentile of intensities for negative control probes, except for the ORF+ skin lesion sample that was analyzed using the 95th percentile.
