mRNA Profiling of miR-17 family inhibition using TuD lentiviral vector in Hep3B hepatocellular carcinoma cell line [array]

To functionally characterize the role of miR-17 family in HCC, lentiviral vector-based miR inhibitor TuD was used to inhibit miR-17 family of microRNAs in Hep3B cell line Methods: Hep3B HCC cell line was acquired from American Type Culture Collection (ATCC) and miR-17 TuD or NC TuD expressing lines were generated. Microarray profiling of miR-17 TuD or NC TuD expressing samples was performed using Affymetrix HG-U133Plus2 arrays. Briefly, starting with 1 ug total RNA, amplified biotin-labeled cRNA targets were produced using the Enzo Target Labeling method. Fragmented, biotin-labeled cRNA was hybridized to the Affymetrix GeneChip® HG-U133Plus2 Genome Arrays overnight at 45C for 16 to 18 hours according to the manufacturer’s recommendations. Stain, wash (GeneChip Fluidics Station 450, EukGE-WS2v5_450 script) and scan (GCS 3000 7G with the GeneChip® AutoLoader) were performed according to the manufacturer’s recommendations. Microarray data were GC Robust Multiarray Average (GCRMA) normalized using R and the bioconductor.org package gcrma and Log2 transformed. Gene expression profiles for TuD expressing samples (triplicate) and NC TuD expressing samples (triplicate) were generated using Affymetrix HG-U133Plus2 microarrays