Humanht 12 microarray platform

Patients were treated by standard therapy including surgery and chemo- or radiation therapy. We used Kaplan-Meier analysis (log-rank test) to compare subjects with high (≥ median) versus low (< median) level of expression by overall survival. Each gene was analyzed separately.
To test relevance of the results of the analysis of primary melanomas to metastatic melanomas, we identified differentially expressed genes between LN-positive versus LN-negative primary tumors. A total of 14 genes that were significant in both InterMEL and TCGA samples have been analyzed: PIK3CG, PLCG2, TLR4, CASP8, ITGA8, IL2RB, FLT1, RAC2, NTRK1, IL2RA, IL7R, JAK2, RASGRP1, and HSP90B1. All these genes were downregulated in LN-positive samples. The logarithm of the average expression across the genes was used as a score to predict survival. The median score was used to stratify cases into high or low average expression. Metastatic (total 460) TCGA samples and metastatic (total 214) samples from the GEO GSE65904 dataset [41 (link)] were used in the analysis. In GSE65904 gene expression was measured by the Illumina HumanHT-12 microarray platform (48,107 probes). Probe expressions were converted into gene expressions by choosing the probe with the largest average expression across all samples.

T47D and MDA-MB-231 cells were transfected with control or BRCA1P1-ASOs, incubated for 24 hours, and processed for RNA purification. MDA-MB-231 cells with BRCA1P1-knockout or wild type genotype were also subjected to RNA purification. Total RNAs were isolated with the RNeasy kit (Qiagen, Valencia, CA) following the protocol provided by the manufacturer. To avoid any possibility of DNA contamination, total RNAs were treated with RNase-free DNase I (Qiagen, Valencia, CA). Quality control was performed with the Agilent 4200 TapeStation system (Agilent Technologies, Santa Clara, CA). RNAs with RNA integrity numbers greater than eight were selected and subjected to the Illumina Human HT12 microarray platform. The microarray data were analyzed by Significance Analysis of Microarrays (SAM) between control or BRCA1P1-ASO-treated cells or between BRCA1P1-knockout and wild type cells. The significant gene expression in ASO-treated cells in microarray experiments was identified and subjected to DAVID Functional Annotation Bioinformatics Microarray Analysis. The microarray datasets are available through GEO (GSE112572 & GSE112573).