Lo et al. BMC Cancer 2012, 12:235 http://www.biomedcentral.com/1471-2407/12/235 UT) by agitating the hybridization solution for 50 hours at 50°C. The arrays were scanned using scanner Gene- Pix4000B (Axon Instruments, Foster City, CA) and the images were segmented and transformed to Cy3-Cy5 log ratio using GenePix software (Axon Instruments, Inc). Further data processing, including normalization and data analysis was performed and is described in detail in the Result section. Biological software analysis for candidate gene selection The array results were further analyzed by MetaCore™ analysis software (GeneGo, Inc., St. Joseph, MI) to iden- tify genes involved in cellular pathways, disease-specific biomarker genes, and genes annotated in the gene ontol- ogy databases. Calculation of statistical significance throughout MetaCore was based on P value, calculated based on hypergeometric distribution [14]. P value of 0.05 was used for the cutoff of candidate gene selection. Real-time quantitative polymerase chain reaction (qPCR) qPCR was used to confirm the array-CGH results and to determine the DNA copy number variations of identified candidate genes in 30 NSCLC tumor and corresponding normal samples from Asian, and 30 NSCLC tumor and 12 autopsy samples from Caucasian. DNA copy number was quantified by qPCR using the ABI 7900 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Genomic DNA (100 ng) was amplified using the SYBR Green PCR Master Mix (Roche, Palo Alto, CA) and specific primers for each candidate gene located in exon and adjacent intron, and the GAPDH internal con- trol gene located within the promoter region. The pri- mer sequences and annealing temperature are listed in the Additional file 4: Table S3. The mean DNA dosage ratio between each candidate gene and the GAPDH in- ternal control gene was calculated in all normal and all tumor samples analyzed [15]. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) RT-qPCR was used to measure the mRNA expression of four candidate genes in 45 NSCLC tumor and the corre- sponding normal samples from Asian, and 24 tumor and corresponding normal samples from Caucasian (Tis- sueScan Lung Cancer Panel IV: HLRT304; OriGene, Rockville, MD). RT-qPCR was conducted using the ABI 7900 Sequence Detection System (PE Applied Biosys- tems). Total RNA (4 μg) from tumor and the corre- sponding normal tissues were reverse transcribed into cDNA and amplified using the SYBR Green PCR Master Mix (Roche) and specific primers for each candidate gene and the ß-actin internal control gene. The primer sequences and annealing temperature are listed in the Additional file 4: Table S3. The mean mRNA expression Page 3 of 13 ratio between each candidate gene and the ß-actin in- ternal control gene was calculated in all normal and all tumor samples analyzed. Chromogenic in situ hybridization (CISH) CISH was done on 4 μm thick formalin-fixed paraffin embedded tissue section slides following the standard protocol described in Chang et al. [16]. The probes were digoxigenin-labeled with BAC DNA (RP11-375N9 and RP11-170O19 for ARHGAP19 gene probe; RP11-391k6 for ZNF322A gene probe), and then ethanol precipitated with 10 μg Human Cot-1 DNA. At least 100 non- overlapping and intact tumor nuclei were evaluated. The tumor was considered as CISH amplification positive if the copy number was ≥4 signals per nucleus in more than 40% of tumor cells evaluated [16]. Immunohistochemistry (IHC) The protein expression level of the candidate gene, PAFAH1B1, was evaluated by IHC in 28 NSCLC samples from Asian and 37 lung cancer samples from Caucasian tissue array. Paraffin blocks of tumors were cut into 5-μm slices and then processed using standard deparaffinization and rehydration techniques. Polyclonal antibody against PAFAH1B1 (1:800; Novus, Littleton, CO) was used to detect the protein expression. The evaluation of IHC was conducted blindly without prior knowledge of the clinical and pathologic characteristics of the cases. The samples were graded high expression when >50% tumor cells were stained positive using adequate staining in surrounding normal stromal and epithelial cells. Statistical analysis The Bayes regression approach was used to determine the chromosomal breaking points [17]. Candidate gene selection by MetaCore (GeneGo, Inc.) was calculated based on hypergeometric distribution [14] and a P value of 0.05 was used for the cutoff. A two-tailed t test was used to determine the statistical significance of differ- ence in DNA dosage and mRNA expression level of can- didate genes in clinical samples. Chi-square test was conducted to examine the association between overex- pression of candidate genes and clinical pathological parameters, including sex, age, tumor type, tumor stage, and smoking. A P < 0.05 was considered to be statisti- cally significant. Results Generation of a specialized non-gapped CGH microarray representing 18 human chromosome imbalance hotspot regions This study constructed a non-gapped BAC array covering 18 human chromosome regions including 2p22.1-p25.1,