patient age at primary diagnosis (years): 35 patient sex: Male patient age at stage iii sample banked (years): 54 survival from stage iii tumour banked (months): 51.62 survival from primary melanoma (months): 284.22 patient last status: Alive NSR number of primary melanomas: 1 stage at primary diagnosis 5th edition: Stage II
Treatment protocol
Tissue samples were homogenized using a high-speed agitation polytron blender (Kinematica, Luzern, Switzerland) in the presence of Trizol.
Growth protocol
Total RNA was extracted from 20-30mg of fresh frozen tissue.
Extracted molecule
total RNA
Extraction protocol
The RNA was isolated and purified with an RNeasy purification kit (Qiagen RNeasy purification kit- Qiagen Pty Ltd., Clifton Hill, Victoria, Australia) with DNAse I digestion on the column. The quality of the RNA preparations was assessed using the Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). RNA integrity scores were >8 for all samples analyzed.
Label
biotin
Label protocol
cRNA amplification and labelling with biotin were performed using Illumina TotalPrep RNA amplification kit according to the manufacturer’s directions (Ambion, Inc., Austin, TX, USA) with 250ng total RNA as input material.
Hybridization protocol
Gene expression analysis was performed using the Sentrix Human-6 v3 Expression BeadChips (Illumina, Inc., San Diego, CA, USA), and BeadStation system from Illumina as per manufacturer’s instructions.
Scan protocol
Gene expression analysis was performed using the Sentrix Human-6 v3 Expression BeadChips (Illumina, Inc., San Diego, CA, USA), and BeadStation system from Illumina as per manufacturer’s instructions.
Data processing
Quality control was performed on all chips using R/Bioconductor and the lumi package (www.bioconductor.org). Data normalization was performed using a variance-stabilizing transform (VST) and quantile normalization as implemented in the lumi package for R/Bioconductor. To reduce false positives, unexpressed genes (detection p-value >0.01) were removed, reducing the number of probes analysed from 48,802 to 26,085.
