Comparison of Cy5-labeled IM-a cells and Cy3-labeled reference. 40 microgram of total RNA was used.The quality of extracted RNA was checked using a 2100 Bioanalyzer. The Label star array kit (Qiagen) was used for cDNA labeling with modification.EBV specific primers and control gene specific primers were used instead of oligo-dT. Prehybridization was done in buffer containing 5XSSC, 0.1% SDS, and 1% BSA for 1hr. Hybridization was done using Hybridization buffer (Sigma-Aldrich co) containing 1/100 vol. of Salmon testis, at 42oC for 16 hrs. Washing was done with washing buffer containing 2 x SSC, 0.1% SDS for 5 min with buffer containing 0.5 x SSC, 0.1 % SDS for 3 min,followed by buffer containing 0.5% SSC for 3 min, at room temp. Scanning of array data: GenePix 4000B. Data analysis was done using a Genomic Profiler (http://bio.mki.co.jp/en/product/index.html). Normalization: by signals obtained from 4 human house keeping genes. This DNA chip aimed to investigate the viral gene expression in EBV positive cell lines which is not be expressed in the reference cells. Therefore, we scanned the hybridization signals by GenePix 4000B as raw data, and then converted data into Genomic Profiler software to normalize the ratio of Cy5-labeled sample and Cy3-labeled reference properly. All the signals from viral genes were normalized by signals obtained from 4 housekeeping genes whose expression should be equal in both samples and the reference. In general, the signal of Cy-3 labeled cDNA from reference cells are stronger than that of Cy5-labeld cDNA from infected cells, therefore, this procedure was extremely important to normalize the amount of applied cDNA for each microarray. Detailed protocol of microarray analysis of virus gene: Ohyashiki JH et al. Biochem Biophys Res Commun. 2005 329(1):11-17 Keywords = EBV
