Gene expression profile of 293T cells transfected with DOCK10

The goal of this study was to identify changes in the gene expression profile (GEP) of 293T cells in response to transfection with DOCK10 during 24 hours. Human embryonic kidney 293T cells, highly transfectable fibroblastoid cells growing adherently as monolayers, were cultured in Dulbecco’s MEM supplemented with 10 % FCS, 50 U/ml penicillin, 50 U/ml streptomycin, 2,5 µg/ml amphotericin B, and 2 mM L-glutamine. Cells were transfected at subconfluency with pSG5-HA-DOCK10.1 plasmid or empty pSG5 plasmid using lipofectamine, and cultured for 24 hr. Following detachment of cells with 0.05%-EDTA 0.02% in PBS, total RNA was isolated using the miRNeasy Mini Kit (Qiagen) and labeled with cyanine 5-CTP. Total RNA isolated from untransfected 293T cells was labelled with cyanine 3-CTP and used as reference sample. Both 293T samples (pSG5 and DOCK10), were labelled with cy5 and used as test samples. The reference sample and the test samples were hybridized together onto Agilent Whole Human Genome Microarrays (4×44K) targeting 19,596 Entrez Gene RNAs (technology 014850), scanned in an Agilent G2565CA DNA Microarray Scanner using the Agilent Scan Control software. Images were analyzed with the Agilent Feature Extraction software, which computes log ratios (test vs reference) following normalization correction by linear and Lowess methods.