Unknown,Transcriptomics,Genomics,Proteomics

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Effects of EDAG knockdown on gene expression profiles in human erythroid cells

ABSTRACT: To investigate the effects of EDAG knockdown on gene exrpession profile in human cord blood CD34+ cells with EPO treatment with microarray assay. Freshly isolated human cord blood CD34+ cells were pre-activated for 24 hours and then infected with EDAG RNAi lentivirus or control lentivirus for 3 times within 24 hours. Then the cells were cultured in the presence of EPO to induce erythroid differentiation. Four days later, the cells were harvested for microarray test. The microarray showed that 3149 differentially expressed genes in siEDAG group compared to control group.The altered genes were associated with gene exrpression, cell cycle and hematopoietic differentiation. These data show that EDAG knckdown led to down-regulation of erythroid genes which are activated by GATA1. Human CD34+ cells infected with EDAG RNAi or control lentivirus were treated with EPO (5U/ml) for 4 days and gene expression were measured using Agilent human whole Genome 8*60K array. Two replicates.

ORGANISM(S): Homo sapiens

SUBMITTER: wang liang

PROVIDER: E-GEOD-46720 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mononuclear cells were isolated from umbilical cord blood (UCB) using Lymphoprep sucrose-gradient centrifugation (1.077 g/ml, Nycomed Pharma, Oslo, Norway). Immunomagnetic cell separation, using magnetic beads coated with CD34 antibodies (Miltenyi Biotec, Gladbach, Germany), was performed to isolate CD34-positive hematopoietic progenitor cells (CB-CD34+ cells). To generate retrovirus, bicistronic retroviral DNA constructs were used expressing the ETV6-RUNX1 or AML1-ETO fusion gene and enhanced Green Fluorescent Protein (eGFP). As a control, a construct expressing only eGFP was used. Both vectors consisted of a pMSCV promoter region, an internal ribosomal entry site and an ampicillin resistance cassette. HEK293T cells were co-transfected with these constructs and second-generation retroviral packaging vectors using XtremeGENE 9 tranfection reagents (Roche, Basel, Switzerland). Viral particles were collected in IMDM 48 hours after transfection. CD34+ haematopoietic progenitors, pre-cultured overnight as described above, upon which cells were divided in two fractions. One fraction was transduced with ETV6-RUNX1-IRES-eGFP or AML1-ETO-IRES-eGFP, while the other fraction was transduced with control EV-IRES-eGFP. Transductions were performed with fresh retrovirus in retronectin (Takara, Otsu, Japan) coated wells. After sorting, DAPI- CD34+ GFP+ CB-CD34+ cells were lysed and RNA was extracted using Nucleospin RNA XS extraction columns according to manufacturer’s protocol (Macherey-Nagel, Düren, Germany). Quality of RNA was determined by on-chip-electrophoresis using a RNA Pico Chip according to manufacturer’s protocol (Agilent Technologies, Santa Clara, CA, USA). RNA Integrity scores (RIN) were higher than 8 for all samples. RNA was subsequently linearly amplified using the Nugen WT-Amplification™ pico system (Nugen, San Carlos, CA, USA). This system is based on RNA-dependent DNA polymerase activity and was previously reported to be most suitable for amplification and gene expression of picograms of input RNA.

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Effects of EDAG knockdown on gene expression profiles in human erythroid cells

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