Alterations in total and in polysomal mRNA fraction in response to expression of RNA binding motif protein 35A. Homo sapiens

The gene coding for RNA binding motif protein 35A (RBM35A) is inactivated by frameshift mutations in an LS180 colon carcinoma cell line and in approximately in 50% of colon tumors with microsatellite instability. To get insight into the mechanism of action of these putative tumor suppressor gene we expressed functional copy of the RBM35A cDNA in the LS180 cells. We analyzed alterations in mRNA profiles in total and in polysomal fraction of mRNA in LS180 cells in response to expressing RBM35A gene under Tet off tetracycline inducible promoter. Overall design: Tet-off LS180-RBM35A cells with restored expression of RBM35A protein (incubated without Dox for at least six days) and their parental cells maintained in the presence of Dox were grown to no more than 80% confluency to ensure good loading of polysomes. Total RNA was extracted using TRIZOL reagent (Invitrogen) according to manufacturer instructions. Polysomal fractions of mRNA of cells expressing or not expressing RBM35A protein were isolated using sucrose density gradient sedimentation. Ten micrograms of polysomal RNA from RBM35+ and RBM35- cells were used to synthesize double-stranded cDNA using a SuperScript II reverse transcriptase kit (Invitrogen). Biotinylated cRNA was synthesized from double-stranded cDNA using the Enzo Bioarray High Yield transcript labeling kit (EnzoLife Sciences), then purified using the GeneChip sample cleanup module (Affymetrix) and fragmented. Fragmented cRNA was hybridized to Affymetrix HG-U95Av2 GeneChips.