Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human CD34+ treated with EPO at day 7 and 14 to investigate the effect of EPO on erythroid maturation.

ABSTRACT: To study transcriptional profiles in human CD34+ cell in EPO condition on day 7 and day 14. Experiment Overall Design: After obtaining informed consent, human CD34+ cells were isolated in high purity from the peripheral blood of normal human volunteers. The cells were cultured at a concentration of 104 -105 cells/mL in medium supplemented with 4 U/mL EPO (Amgen, Thousand Oaks, CA) as described previously.1 Cells were enumerated using an electronic cell counter (Coulter, Hialeah, FL). Adult and fetal hemoglobin was analysed by HPLC. RNA was extracted using QIAshredder and Rneasy Minikit.

ORGANISM(S): Homo sapiens

SUBMITTER: Jeffery Lynn Miller

PROVIDER: E-GEOD-7874 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Hypoxic-ischemic (HI) injury in the developing brain is a common cause of disability in children, and there are no effective treatments at this time. Erythropoietin (EPO) has recently gained interest as a neuroprotective drug, and EPO and its receptor are expressed within the central nervous system. We have recently shown that pretreatment with EPO markedly reduced brain injury caused by unilateral hypoxic-ischemic insult in 7 day old mice. EPO did not reduce early signs of neuronal injury at 6 hours, but significantly protected the neonatal brain when assessed 24 hours and 7 days after HI. The mechanism of this delayed protection is unclear, but is thought to involve transcription of neuroprotective genes, possibly subsequent to activation of NFkB. By comparing gene expression in EPO- and vehicle- (VEH) treated mice after HI, we should gain insight into the mechanisms underlying the neuroprotective effects of EPO and may identify additional targets for therapeutic interventions. We will compare gene expression patterns in 7-day old mouse forebrain after 1) VEH pretreatment plus sham surgery, 2) VEH pretreatment plus HI, and 3) EPO pretreatment plus HI. We will thus identify genes induced by HI in the developing brain and characterize changes in gene expression caused by EPO pretreatment. We will use the model of neonatal hypoxic-ischemic injury and EPO treatment parameters that were used in our prior studies demonstrating neuroprotection. Based on the time-course of neuroprotection defined in our prior study and the pharmacokinetic profile of EPO, we will examine gene expression 18 hours after HI. We hypothesize that changes in gene expression after EPO pretreatment underlie the neuroprotective effects of this cytokine after neonatal hypoxic ischemic injury. We will examine gene expression in 3 groups: 1) 1) VEH pretreatment + sham surgery, 2) VEH pretreatment + HI, and 3) EPO pretreatment + HI. EPO (5U/g, i.p.) or VEH was injected in 7 day old mice, drawn from 4 litters, with 3 to 4 pups per treatment group in each litter. One hour later, the right common carotid artery was ligated under isoflurane anesthesia, animals were allowed to recover for 90 min and were then placed in hypoxic chambers (10% oxygen, balance nitrogen) for 50 min. The animals subjected to sham surgery received isoflurane anesthesia for a comparable period, incision and dissection to visualize the common carotid artery, but no ligation and no hypoxia. Eighteen hrs later, animals were anesthetized with isoflurane and the right hemisphere was rapidly dissected and placed in RNA Later (Qiagen) at 4C. Total RNA was isolated using an RNeasy lipid tissue mini kit (QIAzol lysis and RNeasy purification, Qiagen). RNA concentrations were determined spectrophotometrically and an aliquot of each sample was examined by gel electrophoresis to screen for degradation. Samples were stored at -80C. After quality control screening, we selected 5 male and 5 female samples per treatment group (extra samples were reserved). We plan to pool 1 male and 1 female for each microarray sample and we plan to run 5 microarrays from each treatment group, for a total of 15 microarrays. We would like to have Agilent Bioanalyzer quality control assays on the individual samples carried out by the consortium before pooling. We will send 10 micrograms of each sample for Agilent QC and subsequent pooling of equimolar amounts. We would like to use Affymetrix mouse gene chips (please advise which specific chips are available; are you using the GeneChip Mouse Expression array 430A or the GeneChip Mouse Genome 430 2.0 Array?).

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Transcription profiling of human CD34+ treated with EPO at day 7 and 14 to investigate the effect of EPO on erythroid maturation.

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