3000 7g scanner

Preparation of cRNA, hybridization, and scanning of microarrays was performed according to the manufacturer’s protocol (Affymetrix, Thermo-Fischer, Santa Clara, Ca, USA). In brief, 300 ng of total RNA extracted from homogenised patient samples isolated using Trizol was converted into double-stranded cDNA by reverse transcription. Biotin-labelled cRNA was generated by converting the cDNA sample using the Genechip WT plus reagent kit (Affymetrix, Thermo-Fischer, Santa Clara, Ca, USA). Labelled cRNA was hybridized to the Affymetrix GeneChip^® Human Gene 2.0 ST Array while rotating at 60 rpm for 16 h at 45 °C. After hybridization, the microarray was washed using the Affymetrix Fluidics Station according to the manufacturer’s protocol. The chips were scanned in an Affymetrix 3000 7G scanner.
Differential gene analysis expression was carried out using Applied Biosystems Transcriptome Analysis Console (TAC) software 4.0.2. Resulting gene lists were filtered for +/- 2-fold changes, a p-value < 0.05 and an overall FDR F-Test: <0.005.

RNA-Seq: Biological replicates were sequenced at the Core Facility for High-Throughput Genetics and Genomics, located at the Medical Faculty, Westfälische Wilhelms-Universität Münster. For all samples, the RNA integrity number (RIN) was measured. The four samples per condition with the highest RIN were selected. Poly-A-enriched, strand-specific antisense RNA sequences were applied on 1000 ng RNA that had been PCR-amplified for 8 cycles. The abovementioned samples were sequenced at a read depth of 25 × 106, involving 75 cycles. Demultiplexed fasta files were further analyzed at UZH.
Microarray: The fragmented and biotinylated RNA samples were prepared according to the standard Affymetrix WT PLUS Reagent Kit protocol (Affymetrix GeneChip^® WT PLUS Reagent Kit, 902280) from 100 ng total RNA starting material and 5.5 μg cDNA intermediate product. DNA targets were hybridized for 17 h at 45 °C on GeneChip Human Transcriptome Arrays 2.0. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 according to the standard GeneChip Expression Wash, Stain and Scan protocol (Affymetrix GeneChip Wash, Stain and Scan Kit, 900720). Subsequently, the GeneChips were scanned using the Affymetrix 3000 7 G scanner. Raw CEL files were further analyzed at UZH.

All patient specimens collected at the time of surgery were split into tissue for pathology and tissue for the biobank. Tissue for the biobank was embedded in Tissue-Tek® O.C.T™ Compound and snap frozen in liquid nitrogen before storage at -80°C. Sections were examined by a genitourinary pathologist to identify carcinoma cell content. Following, cresyl violet stained tissue was microdissected using the PALM laser microbeam system. RNA extraction was performed using RNeasy Micro Kits (Qiagen) according to manufacturer protocols. RNA quality was assessed using an Agilent Bioanalyzer 2100 (RIN: 2.4-8.8; median 5.9). Total RNA was amplified and converted to cDNA using Nugen Pico-RNA system. The two-round amplification kit is optimized to amplify low volumes and poor quality RNA for Affymetrix array analysis. After amplification, the cDNA was fragmented and labeled using NuGen FL-Ovation kit, loaded onto the Affymetrix U133 Plus 2.0 Gene Chip according to the manufacturer’s protocol, and scanned using the Affymetrix 3000 7G Scanner.

cRNA was prepared with GeneChip® 3’ IVT PLUS Reagent Kit (Applied Biosystems, Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. The GeneChip Drosophila Genome 2.0 Arrays (Applied Biosystems, Thermo Fisher Scientific Inc.) were hybridized, stained, and washed according to the manufacturer's protocol and scanned with Affymetrix 3,000 7G scanner. The data set includes the scans of six GeneChip™ Drosophila Genome 2.0 Arrays (designed using Dmel Release 3.1), representing of Canton‐S or D18 lanes in three independent biological repeats. All data were submitted to NCBI GEO database, under accession number GSE146725.
CEL files obtained after scanning were analyzed using the Transcriptome Analysis Console software version 4.0.2 (reference Drosophila melanogaster genome release 3.1). Background correction and normalization were made by RMA algorithm using default parameters. A probe was selected to be differentially expressed if its p‐value was < .01 and its mean fold change value across three replicates was more than twofold. Gene ontology analysis for enrichment of biological processes was done by DAVID version 6.8 (data base of all annotated genes was used as a background list, enrichment cut off p < .1), up‐ and downregulated genes were analyzed separately. In addition, for searching GO terms for individual genes FlyBase was used.

Samples (derived from SOX10-GFP hES cells) were prepared according to the Affymetrix WT Plus protocol for Gene Chip Whole Transcript Expression Arrays. Briefly 200 ng of high quality total RNA, (RNA integrity number (RIN) greater than 9), was converted to double stranded cDNA with the introduction of a T7 polymerase binding site. This allowed the synthesis of an antisense RNA molecule against which a sense DNA strand was prepared. The RNA strand was digested and the resulting single stranded DNA fragmented and biotin labelled. Along with appropriate controls the labelled fragmented DNA was hybridised to Affymetrix Clariom D arrays overnight using the Affymetrix 640 hybridisation oven; 16 hr with rotation at 60 rpm at 45°C. The arrays were washed and stained according to standard protocols which allowed the introduction of streptavidin-phycoerythrin in order to generate a fluorescent signal from the hybridised biotinylated fragments. The washed and stained arrays were scanned using the Affymetrix 3000 7G scanner with autoloader. The generated CEL files were taken forward for analysis.