Computational protocol: Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples

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Protocol publication

[…] Codelink® Whole Mouse Genome Bioarrays (GE/Amersham) of one sample of WT, D6, D54, WT/D6, WT/D54 and MIGR NIH3T3 cells cultivated in the three different conditions SCF+, SCF− and imatinib were used for the analysis of differential gene expression. These microarrays contain 3.3×104 single-stranded 30-mer oligonucleotide probes for mouse genes and transcribed sequences.For the experiment, the various NIH3T3 KIT expressing cell lines were seed at 1 million per 6 cm plate in complete medium without SCF and keep in this condition during one night (16 hours). Then, the cells were incubated for 24 hours with or without rhSCF and/or imatinib as indicated.Before RNA extraction, cells were detached following a classical trypsine protocol (Invitrogen®), washed two times in cold PBS (Invitrogen®) and finally freezed at −80°C without supernatant.Total RNA of NIH3T3 cells were extracted according to the manufacturer’s protocol using TRIzol® method (Invitrogen®, Carlsbad, CA). RNA quality was controlled by spectophotometry using ND-1000 Nanodrop® and by microchips on Agilent bioanalyzer 2100 (Agilent® Technologies, Palo Alto, CA, USA). Then, for high density microarrays, total RNA (100 ng) were amplified and biotin-labeled by in vitro transcription (IVT) with a MessageAmp™ II-Bacteria RNA Amplification Kit (Ambion/Applied Biosystem®). Before amplification, spikes of synthetic mRNA at different concentrations were added to all samples; these positive controls were used to ascertain the quality of the process.Ten micrograms of biotinylated cRNA was fragmented and hybridized onto Codelink® Whole Mouse Genome Bioarrays (GE/Amersham) for 18 hours at 37°C at 300 rpm in an incubator. The slides were washed and stained with streptavidin-cy5 (GE Healthcare) according to the manufacturer’s protocol. The slides were scanned using a Genepix® 4000B scanner (Axon, Union City, USA) and Genepix® software, with the laser set at 635 mm, the laser power at 100%, and the photomultiplier tube voltage at 60%.The scanned image files were analyzed using CodeLink® expression software, version 4.2 (GE Healthcare), which produces both a raw and normalized hybridization signal for each spot on the array. The relative intensity of the raw hybridization signal on arrays varies in different experiments. CodeLink® software was therefore used to normalize the raw hybridization signal on each array to the median of the array (median intensity is 1 after normalization) for better cross-array comparison. The threshold of detection was calculated using the normalized signal intensity of the 100 negative control samples in the array; spots with signal intensities below this threshold are referred to as “absent”. The complete set of raw and normalized data is available at the GEO database under accession number GSE33968.Selections of genes were performed by comparisons between group1 and group2 or group1 and group3 … Each sample from one group was compared with each sample from others groups, and only genes showing a variation of average fold change ≥2 were retained. A gene was considered only if it met the above criteria in all comparisons and if the detected signal was above the background for at least one of the compared tumor groups, thereby carrying a statistically significant absolute call ‘present’ or ‘marginal’ in all samples. The retained genes of interest were listed and classified according to their biological functions on the basis of the Gene Ontology Consortium using Ingenuity® Pathway Analysis (Mountain View, CA). […]

Pipeline specifications

Software tools Codelink, IPA
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Neoplasms, Machado-Joseph Disease