Genomestudio gene expression module

Purified total RNA from both early and late progression tumours derived from each 143B cell line was labelled, amplified and hybridized to HumanHT-12 v4 Expression BeadChip Kit (Illumina, Scoresby, VIC, Australia) in triplicate though the Australian Genome Research Facility Ltd (AGRF; Walter Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia), according to the manufacturer’s protocols. Expression data were analysed using GenomeStudio Gene Expression Module (Illumina) and the limma package of R Bio-conductor⁵⁰ (http://www.r-project.org).

Whole genome gene expression analysis was performed using the Illumina (San Diego, CA, USA) WG-DASL assay according to manufacturer's protocol. Briefly, 100 ng of FFPE RNA was converted to cDNA by the WG-DASL assay using biotinylated-tagged random nonamer and oligo (dT) primers. The biontinylated cDNA was then mounted onto a streptavidin-coated support and further extended and ligated by gene-specific oligonucleotides (DAP). Subsequently, PCR amplification was performed. The resulting PCR products were eluted and hybridized to the Illumina Human-Ref v3.0 Beadchip and scanned with the Illumina iScan Reader. The image intensity values from the microarray images generated were then analysed by the GenomeStudio Gene Expression Module (Illumina, San Diego, CA, USA) software. The processed methylation values were subsequently used for further analysis in this study.

For gene expression analyses, we used human hippocampal biopsy tissue from patients with hippocampal sclerosis (n=79) versus patients with lesion-associated (low-grade neoplasms or dysplasia; n=35) chronic TLE, who underwent surgical treatment in the Epilepsy Surgery Program at the University of Bonn Medical Center due to pharmacoresistance. In all patients, presurgical evaluation using a combination of non-invasive and invasive procedures revealed that seizures originated in the mesial temporal lobe42 (link). All procedures were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Bonn Medical Center. Informed written consent was obtained from all patients. Clinical characteristics per subgroup are described in Supplementary Table 3. mRNA analyses for Ca_V3.2 and MTF1 were carried out analogous to a procedure described elsewhere in detail43 (link). Briefly, RNA from biopsies representing all hippocampal subfields served to generate 750 ng cRNA used for hybridization on Human HT-12 v3 Expression BeadChips with Illumina Direct Hybridization Assay Kit (Illumina, San Diego, CA) according to standard procedures. We extracted data for Ca_V3.2 and MTF1 analysed by Illumina's GenomeStudio Gene Expression Module and normalized using Illumina BeadStudio software suite by quantile normalization with background subtraction.