Computational protocol: MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells

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Protocol publication

[…] MCF-7 cells were treated with 10 μM MELK-T1 for 48 h. Cells were lysed using RLT (RNeasy lysis) buffer (Qiagen) and RNA was extracted with the RNeasy 96 kit (Qiagen). All microarray-related steps for target preparation, including the amplification of total RNA and labelling, were carried out as described in the GeneChip®3′ IVT Express Kit User Manual (Affymetrix 2004). Biotin-labelled target samples were hybridized to GeneChip® Human Genome HT U133 PLUS PM 96-Array containing probes for approximately 19000 genes. Target hybridization was processed on the GeneTitan® Instrument according to the instructions provided in the User Guide for Expression Array Plates. Images were analysed using the GeneChip® Command Console Software (AGCC; Affymetrix). All data were processed using the statistical computing R-program (R version 3.0.1; R Development Core Team) and Bioconductor tools []. The gene expression values were computed using RMA (robust multi-array average) []. Grouping of the individual probes into gene-specific probe sets was performed based on Entrez Gene using the metadata package hthgu133pluspmhsentrezg (version 15.1.0 []). The resulting log2-transformed data were the basis for differential expression analysis using Significance Analysis of Microarrays software []. The affected pathways were analysed using MLP (mean log P analysis) [] and IPA (Ingenuity Pathway Analysis; Ingenuity Systems, www.ingenuity.com). The data have been submitted in GEO (gene expression omnibus) (GSE62477). […]

Pipeline specifications

Software tools SAM, IPA
Databases GEO Gene
Application Gene expression microarray analysis
Diseases Breast Neoplasms, Neoplasms