Computational protocol: Anti-tumour activity of two novel compounds in cisplatin-resistant testicular germ cell cancer

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Protocol publication

[…] DNA microarray analysis and RT-PCR (including primer and incubation times) were performed as described previously (). For microarray analysis in brief, total cellular RNA was extracted from cells using ArrayGrade Total RNA Isolation Kit (SABiosciences, Frederick, MD, USA). RNA concentration was measured by absorption spectrophotometry (GeneQuant, Biochrom, Cambridge, UK). Using the True-Labeling AMP 2.0 amplification kit (SABiosciences), the mRNA was reversely transcribed into cDNA and converted to biotin-labelled cRNA using biotin-16-UTP (Roche, Mannheim, Germany) by in vitro transcription. The cRNA samples were purified with an ArrayGrade cRNA cleanup kit (SABiosciences). Thereafter, the probes were hybridised to the pretreated Oligo GEArray Human Toxicology and Drug Resistance Microarray (OHS-401, SABiosciences). After several washing steps, array spots binding cRNA were detected by chemiluminescence staining. Image acquisition was performed using X-ray films and a digital scanner. Spots were analysed and converted to numerical data by using the GEArray Expression Analysis Suite software (SABiosciences). Data evaluation included background correction (subtraction of minimum value) and normalisation to reference genes. The cut-off value for upregulation was set at a 1.5-fold increase of the ratio of genes in the treated samples, whereas downregulation was set at a 0.5-fold expression of genes in the treated samples.Gene ontology (GO) analysis of differentially expressed genes with a focus on biological processes was performed by using DAVID software tool (, ). Venn diagrams were created using Venny software tool (; […]

Pipeline specifications

Software tools DAVID, VENNY
Application Gene expression microarray analysis
Organisms Gallus gallus, Homo sapiens
Diseases Neoplasms
Chemicals Cisplatin, Platinum