The Effect of RBM5 Gene Manipulation on Expression Data from Day In Vitro 6 Primary Rat Cortical Neurons

RNA Binding Motif 5 (RBM5) is a nuclear splicing factor. Prior studies show that RBM5 overexpression in cancer cells alters gene splicing, gene expression, and induces cell death and/or growth arrest. The role of RBM5 in primary neurons is unknown, and its potential mRNA targets have not been identified. Using lentivirus based approaches we tested if RBM5 knockdown (i.e. by shRNA) or RBM5 overexpression (DDK-tagged rat open reading frame) alters whole genome expression in primary rat cortical neurons. We used microarrays to examine genes that are up-regulated vs. down-regulation in cortical neurons after RBM5 knockdown vs. overexpression. Brain cortices were collected from Sprague Dawley rat embryos. Primary rat cortical neurons were cultured and maintained using Neurobasal media+B27 supplement. On day in vitro (DIV) zero (i.e. on the day of plating) neurons were transduced with lentivirus. The vector treatment groups were as follows: Group 1: Non-Targeting(NT)/scrambled shRNA control with green fluorescent protein (GFP), Group 2: RBM5 targeting shRNA with green fluorescent protein (GFP), Group 3: Empty vector control, Group 4: DDK-Tagged rat RBM5 open reading frame overexpression vector. For control vs. treatment comparision groups 1 vs. 2, three biological replicates (per group) were done (i.e. different neuron cultures established from different timed pregnant rats on different days). For control vs. treatment comparision groups 3 vs. 4, four biological replicates (per group) were done. (i.e. different neuron cultures established from different timed pregnant rats on different days). Neurons were harvested on DIV6 with TRIzol for total RNA extraction. Reverse transcription was performed on 100ng total RNA and dNTP-T7 random primers. Amplified cRNA was made using second strand synthesis and in vitro transcription. cDNA was produced by a subsequent reverse transcription and then fragmented. Fragments were biotin end-labeled in preparation for hybridization to the RTA. The Affymetrix GeneChip® Hybridization, Wash, and Stain kit was used for array processing. 5.2µg biotin-labeled fragmented ss-cDNA was added to 1X hybridization buffer containing oligo B2, hybridization controls, and DMSO. Following incubation at 95°C and 45°C for 5 minutes each, 200µL hybridization mix was loaded onto each array. Arrays were incubated for 16h in a GeneChip 645 hybridization oven (Affymetrix) at 45°C with rotation at 60rpm. Arrays were scanned using the GeneArray® 3000 scanner (Affymetrix). Results were normalized by robust multi-array average (RMA).