RNA expression profiling in G-CCE cells with a knock down of MMTR or overexpression of the full length, N-terminal or C-terminal of MMTR

The goal of this study is to compare total RNA expression profiles among wild type, MMTR knock down, and overexpression of full length, C-terminal and N-terminal in G-CCE cells at d0 and d3 after differentiation. All cell lines were maintained in DMEM supplemented with 15% heat-inactivated fetal bovine serum (FBS; Gibco, 16141079), 1X MEM Non-Essential Amino Acids Solution (Sigma-Aldrich, M7145), 300 μM monothioglycerol (Sigma-Aldrich, M6145), 1X penicillin/streptomycin, and 1000 U/ml Leukima inhibitory factor (LIF, Sigma-Aldrich, ESG1107) in 0.1% gelatin coated cell culture dishes. To induce differentiation of G-CCE cell lines, Leukemia inhibitory factor (LIF) was eliminated from the culture media for 3 days. Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit (Ambion, AMIL1791) to yield biotinylated cRNA according to the manufacturer’s instructions. Briefly, 550 ng of total RNA was reverse-transcribed to cDNA using a T7 oligo(dT) primer.750 ng of labeled cRNA samples were hybridized to each Mouse WG-6 expression v.2 bead array for 16-18 h at 58°C, according to the manufacturer's instructions (Illumina). Detection of array signal was carried out using Amersham fluorolink streptavidin-Cy3 (Invitrogen, SA1010) following the bead array manual. Arrays were scanned with an Illumina bead array Reader confocal scanner Duplicated RNA expression profiles of wild type, MMTR knock down of MMTR or overexpression of the full length, N-terminal or C-terminal of MMTR in G-CCE cells were anlayzed using Illumina BeadStudio v3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis background correction model, variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor.