BioProject

RTqPCR results suggested a relevant role for Pdrg1 in rat hepatoma H35 cells, where its expression was dramatically enhanced. Hence, these cells were chosen as a suitable model for stable Pdrg1 silencing. For this purpose, H35 cells were transfected with appropriate shRNA plasmids against Pdrg1 and stable clones isolated. Among those exhibiting reproducible behavior, clones CN-10 (negative control), 3-44 (shRNA3) and 4-18 (shRNA4) were selected for further analysis. Pdrg1 expression analyzed by RT-qPCR was reduced by 50% and 70% in 3-44 and 4-18 clones, respectively, as compared to CN-10. RNAs of CN-10, 3-44 and 4-18 clones, as well as, RNA of an enriched pool of shRNA3 transiently transfected cells (shRNA3T) were used for expression analysis using Agilent one-color microarrays. Genes exhibiting changes ≥2-fold with FDR<0.05, according to LIMMA analysis, were identified. Pathway analysis was performed with BioProfiling using data of 114 genes (74 upregulated and 40 downregulated) exhibiting similar behavior in the three silenced samples. Selected genes, at least two of pathways with p≤0.02, were used for verification of expression changes by RT-qPCR, together with Pdrg1. Overall design: Silencing of rat Pdrg1 expression was carried out by transfection of rat hepatoma H35 cells with SureSilencing shRNA plasmids (SaBioscience) containing sequences designed for this purpose and a negative control sequence. Stable clones for the negative control (CN), shRNA3 and shRNA4 plasmids were obtained by selection using G418 up to 4 mg/ml. This procedure rendered approximately 200 stable clones for each plasmid, from which one of each was selected [CN-10, shRNA3 (3-44) and shRNA4 (4-18)] for further experiments. Four biological replicates of these stable clones and a transiently transfected shRNA3 H35 enriched pool (shRNA3T), were independently hybridized for each transcriptomic comparison.