cDNA microarray RNA was amplified and biotinylated using the MessageAmp-Biotin Enhanced Kit (Ambion). DNA oligonucleotide probes were synthesized onto a DNA microarray chip called Genopal (Mitsubishi Rayon) in order to detect the 237 genes (200 genes on Chip1 and 37 genes on Chip2) related to the innate immune response. Hybridization was carried out overnight at 65��C using selleck products Genopal in an hybridization buffer[0.12 M Tris-HCl/0.12 M NaCl/0.05% Tween-20]. After hybridization, Genopal was washed with hybridization buffer twice at 65��C for 20 min followed by washing in 0.12 M Tris-HCl/0.12 M NaCl at 65��C for 10 min. Genopal was then labeled with streptavidin-Cy5 (GE Healthcare Bioscience, Tokyo, Japan).
The fluorescent labeled-Genopal was washed for 5 min four times with hybridization buffer at RT and scanned at multiple exposure times ranging from 0 to 40s by DNA microarray reader (Yokogawa Electric Co, Tokyo, Japan). Intensity values with the best exposure condition for each spot were selected. The data presented here have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE20119″,”term_id”:”20119″GSE20119: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=xlmbxyyumcwkeba&acc=”type”:”entrez-geo”,”attrs”:”text”:”GSE20119″,”term_id”:”20119″GSE20119. All data are MIAME compliant, and are also registered with GEO. Statistical analysis To identify the genes that varied significantly among NR, R, SVR and NL groups, one-way ANOVA and Turkey’s post hoc tests were used to assess each of the 237 IFN related-genes on the arrays.
Benjamini-Hochberg correction for multiple hypotheses testing was applied to all tests. P values <0.05 were considered statistically significant. Method of predicting prognosis The patients were randomly divided into two groups: one was used as a TS and the other VS to calculate the prediction discriminant. A prognosis signature (PS) was defined in terms of the expression levels of the six genes that differed significantly between NR and non-NR groups using post hoc analysis (IFI27, IFI44, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), ISG15, MX1, OAS1). A prognosis predictor (PP) was computed by applying a diagonal linear DLDA to the TS [33] and then using it to predict the prognoses of the VS.
The predicted and actual prognoses of VS patients were compared to obtain the following five measures of prognosis prediction performance: accuracy (proportion of correctly predicted prognoses), sensitivity (proportion Brefeldin_A of correctly predicted non-NR), specificity (proportion of correctly predicted NR), PPV (proportion of actual non-NR versus predicted non-NR) and NPV (proportion of actual NR versus predicted NR).
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