Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of gene expression changes in RN2 acute myeloid leukemia (AML) cells at 24hrs and 48hrs after Ddx5 knockdown.

ABSTRACT: Ddx5 inhibition in RN2 cells slows cell proliferation and induces apoptosis within 48-72hrs. The aim of this analysis was to gain insight into how Ddx5 inhibition causes this outcome by analyzing gene expression changes in RN2 cells that occur at early timepoints after Ddx5 inhibition that precedes the timepoint when RN2 proliferation/cell death becomes evident in tissue culture (72hrs after inhibition). Derivatives of RN2 AML cells were prepared that encode doxycycline-inducible expression of either of two different shRNAs targeting Ddx5 (shDdx5.1322 and shDdx5.2086) or a negative control shRNA that targets Renilla Luciferase (shRen.713). Six independent cultures of each derivative RN2 cell line (shDdx5.1322, shDdx5.2086, or shRen.713) were treated with doxycycline at timepoint 0 days to induce expression of the indicated shRNA in the RN2 cells. Each shRNA is co-expressed with dsRed in a doxycycline-induced manner and flow cytometry analysis indicated that doxycycline induced expression of dsRed and the shRNA in 70-to-80% of the cells in each culture. RNA was isolated from three independent cultures of each derivative RN2 cell line at either 24hrs and 48hrs after dpxycycline treatment. Therefore this study consists of 18 samples, sequencing results from biological triplicate samples of RN2-shDdx5.1322, RN2-shDdx5.2086, and RN2-shRen.713 at 24hrs post-doxycycline and sequencing results from biological triplicate samples of RN2-shDdx5.1322, RN2-shDdx5.2086, and shRen.713 at 48hrs post-doxycycline.

ORGANISM(S): Mus musculus

SUBMITTER: Anthony Mazurek

PROVIDER: E-GEOD-53599 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To examine the effect of wtAPE1 and ubiquitin-APE1 fusion proteins on global gene expression. Total RNA from HEK293 derivatives that express vector alone (ctl), wt-APE1, or ubiquitin-APE1 fusion in the presence of doxycycline were analyzed. Three HEK293 derivatives expressing none (vector), wtAPE1, or Ub-APE1 fusion linked at APE1's 24th Lys were incubated with/without doxycycline (dox) 2 Âµg/ml for 16 hr. Each -/+ dox cultures were triplicated. Total RNA was extracted (Qiagen) and then analyzed by LSU's bioinformatics core. The quality and quantity of total RNA was assessed using an ND-1000 Spectrophometer (NanoDrop, Wilmington, DE), NanoChip assay and Bioanalyzer 2100 (Agilent). All samples were of high integrity with RIN # greater than 7 and A260/280 greater than 1.8. Procedures for cDNA synthesis, sense target labeling, and hybridization were carried out as described at http://media.affymetrix.com/support/technical/appnotes/wt_appnote.pdf (Affymetrix, Redwood City, CA). All experiments were performed using GeneChip Exon 1.0 ST Arrays and Whole Transcript Sense Target labeling Assay (version 4, Affymetrix). Overnight hybridization of fragmented single-stranded DNA was carried out in an Affymetrix GeneChip Hybridization Oven 640, then washed and stained with streptavidin-phycoerythrin using GeneChip 450 Microfluidics Station (Affymetrix). Chips were scanned with an Affymetrix High Resolution 3000 (G7) scanner. Signal and background intensities were quantitated by pixel intensity using Affymetrix GeneChip Operating Software (GCOS 1.4). Array quality control assessment was carried out using Robust Multi-chip Analysis (RMA) workflow for Core probesets in Expression Console (Affymetrix). Gene-level analysis for differential gene expression was analyzed in GeneSpring 7.3x (Agilent Technologies) using the sample CHP files. The RMA method was used for the background correction, normalization and average expression measures for the probesets. An expression filter (20th percentile cutoff) was applied to remove genes with low signal intensity values. For differential gene expression analysis, the Welch ANOVA test with asymptotic p-value computation was applied and transcripts with p-value (< 0.05) and fold change (2.0 fold) were selected. Three cell lines (HEK293/pcDNA-FRT empty vector, HEK293/pcDNA-FRT wtAPE1, HEK293/pcDNA-FRT ubAPE1), +/- 2Âµg/ml doxycycline for 16 h. Triplicates of each cell line/condition. Replicates were averaged.

Dataset Information

Analysis of gene expression changes in RN2 acute myeloid leukemia (AML) cells at 24hrs and 48hrs after Ddx5 knockdown.

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