Affymetrix Microarray Protocol for Gene Expression

The microarray study was performed as described in detail elsewhere [30 (link)]. The total RNA isolated from two types of cells was pooled into four samples per group (parental U2OS and ΔMVP-U2OS). The protocol including in vitro transcription, biotin labeling, and cDNA fragmentation was performed using the Affymetrix GeneChip IVT express kit (Affymetrix, Santa Clara, CA, USA). Then, the biotin labeled cDNA were hybridized with the Affymetrix Gene Chip Human Genome U219 microarrays together with appropriate internal controls. The hybridization was performed in the AccuBlockTM digital dry bath hybridization oven (Labnet International, Inc., Edison, NJ, USA) at 45 °C for 16 h. Subsequently, the microarrays were washed and stained by means of the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The microarrays were scanned using the imaging station of the GeneAtlas System. Initial analysis of the scanned microarrays was carried out with Affymetrix GeneAtlas TM Operating Software.