Investigation Design

Investigation Title Ontogeny of erythroid lineage differentiation
Experiment Description Delineate gene anatomy of differentiating primary primitive and definitive RBCs
Experimental Designs development_or_differentiation_design cell_type_comparison_design
Experimental Factor Name Lineage Stage
Experimental Factor Type developmental_stage cell_type
Experimental Factor Term Source REF MGED Ontology MGED Ontology
Person Last Name Palis Kingsley Stoeckert Wang
Person First Name James Paul Chris Qi
Person Email paul_kingsley@urmc.rochester.edu stoeckrt@pcbi.upenn.edu
Person Phone 585-275-5852 215-573-4409
Person Address Box 703, 601 Elmwood Ave., Rochester, NY USA 14642 1415 Blockley Hall, Center for Bioinformatics, 423 Guardian Dr., Philadelphia, PA 19104
Person Affiliation University of Rochester University of Rochester University of Pennsylvania Rutgers
Person Roles investigator submitter investigator investigator
Date of Experiment 2008-05-22
Protocol Name FACS Isolation of Erythroid Cells RNeasy Plus Mini Kit (Qiagen) NuGen Ovation WB Reagent Labeling GeneChip Eukaryotic Target Hybridization Protocol Affymetrix GeneChip Scanner 3000 (GCS3000) gcRMA quantification (bioconductor) Affymetrix MAS 5.0 Probe Cell Analysis
Protocol Description All samples were isolated using FACS.Because primitive erythroid cells mature semi-synchronously in the circulation, we used time as a component for their isolation: 1) proerythroblasts were isolated from e9.5
2) basophilic erythroblasts were isolated from e10.5
3) mix of orthochromatic and polychromatophilic erythroblasts were isolated from e12.5
4) reticulocytes wereisolatedfrom e15.5, using scatter characteristics to purify them from co-occurring definitive fetal reticulocytes. Purity was confirmed using globin specific PCR.
Fetal definitive erythroid cells are produced asynchronously and cells were isolated from e14.5, except for reticulocytes isolated from e15.5.
Bone marrow derived adult erythroid stages were isolated from mature females.
RNA extraction using the Qiagen RNeasy Plus mini kit and gDNA columns (CAT# 74134)including Qiashredder (CAT# 79654) as described in the manufacturer's protocol. Labeling with NuGen ovation Whole Blood Reagent according to manufacturer's protocol and recommendations. The fragmented cRNA is mixed with the hybridization cocktail, and heat shocked at 99C for 5 minutes. In the meanwhile, the probe array is filled with hybridization buffer and incubated at 45C for 10 minutes with rotation. The heated hybridization cocktail is then transferred to a 45C heat block for 5 minutes, and spun at maximum speed in a microcentrifuge for 5 minutes. Fill the probe array cartridge with the clarified hybridization cocktail and place probe array in rotisserie box in 45C oven. Hybridize for 16 hours.
Protocol Parameters Isolation age
Protocol Type fractionate nucleic_acid_extraction labeling hybridization image_acquisition feature_extraction feature_extraction
Term Source Name MGED Ontology
Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php
Term Source Version 1.3.1.1