Wt ovation ffpe system v2

Hierarchical clustering and heatmaps were generated using MultiExperiment Viewer. Statistical significance was calculated using Student’s t-test. GSEA was performed as described (25 (link)). Analysis of human data was performed using cbio-portal.
For the PHS and HPFS analysis, we utilized data from a gene expression profiling study. Briefly, men were sampled from the HPFS and PHS Prostate Tumor Cohort using an extreme case design, which includes 119 men who died of their cancer or developed bony or distant metastases (“lethal”) and 282 men who lived at least 8 years after PCa diagnosis and were not diagnosed with metastases through 2012 (“indolent”). Briefly, RNA was extracted from tissue, then amplified using the WT-Ovation FFPE System V2 (NuGEN), a whole transcriptome amplification system that allows for complete gene expression analysis from archives of FFPE samples. After reverse transcription and fragmentation, the cDNA was hybridized to the Affymetrix GeneChip Human Gene 1.0 ST microarray. After normalization of the data, we mapped gene names to Affymetrix transcript cluster IDs using the NetAffx annotations as implemented in Bioconductor annotation package pd.hugene.1.0.st.v1. We compared the expression of the interstitial genes across the lethal and indolent cases using linear regression.

To conduct the profiling in FFPE tissue, whole transcriptome amplification was paired with microarray technologies. Briefly, RNA samples were extracted on a Biomek FxP automated platform using the Agencourt FormaPure FFPE kit [Cat #A33342] (Beckman Coulter Inc., Brea, CA). The mRNA was amplified using the WT-Ovation FFPE System V2 (Nugen, San Carlos, CA), a whole transcriptome amplification system that allows for complete gene expression analysis from FFPE samples known to harbor small and degraded RNA. Using a combination of 5’ and random primer, reverse transcription created a cDNA/mRNA hybrid. The mRNA was subsequently fragmented, creating binding sites for DNA polymerase. Isothermal strand-displacement, using a proprietary DNA/RNA chimeric SPIA primer, amplified the cDNA. The cDNA was then fragmented and labeled with a terminal deoxynucleotidyl transferase covalently linked to biotin to prepare for microarray hybridization. The labeled cDNA was then hybridized to a GeneChip Human Exon 1.0 ST microarray (Affymetrics, Santa Clara, CA). A pilot study was conducted to validate the reliability and reproducibility of gene expression quantification from FFPE tissues on the study platform (20 (link)).

Whole genome gene expression was measured in tumor and adjacent normal prostate using whole-transcriptome amplification (WT-Ovation FFPE system, v2, NuGEN) paired with microarray technologies (Affymetrix GeneChip HumanGene 1.0ST microarray).^{17 (link)} We processed the data, totaling 20,254 genes from tumor (n=229, of which n=30 were ever statin users) and normal adjacent (n=113, of which n=10 were ever statin users) tissues, as previously described.^{18 (link)} Data are available through the Gene Expression Omnibus (GSE79021).