age: postnatal day 8 tissue: retiina cell type: endothelial
Treatment protocol
Eyes from APB5-treated homozygous Tie2-GFP transgenic mice were enucleated, rinsed with PBS, and kept in staining solution (PBS containing 2mM EDTA, 0.01%, NaN3, 5% fetal calf serum, 50U/ml penicillin, and 0.05mg/ml streptomycin). The retinas were dissected under stereomicroscope, pooled in staining solution, and minced into small pieces. Collected retinal pieces were rinsed twice in PBS and digested in 10 ml of papain/DNase solution (papain (33U/ml; Sigma-Aldrich, St. Louis, MI) in Dulbecco’s PBS (Invitrogen) containing L-cysteine (0.4mg/ml; Nacalai tesque, Kyoto, Japan), EDTA (0.50mM; Sigma), and DNase I (125U/ml; Sigma)) for 60 min at 37°C with gentle agitation. Following digestion, cells were pelleted and treated two times with DNase/ovomucoid solution (a solution containing ovomucoid (2mg/ml; Sigma), DNase I (125 U/ml; Sigma), and BSA (1mg/ml; Wako, Osaka, Japan)) in Dulbecco’s PBS for 60 min at 37°C with gentle agitation. Digested retinal cells were filtered through nylon mesh, centrifuged at 300x g for 5 min to pellet cells, resuspended again with 10ml of DNase/ovomucoid solution incubated at 37°C for 60 min. Cells were rinsed twice in staining solution, pelleted, resuspended with staining solution containing 5µg/ml of propidium iodide, and filtered through nylon mesh. These cells were analyzed and sorted by FACS Aria (Becton Dickinson, San Jose, CA).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using RNeasy Micro-Kit (QIAGEN) and was amplified using MessageAmpII aRNA Kit (Ambion, Austin, TX) to obtain aRNA (>60μg).
Label
biotin
Label protocol
Biotin-labeled cRNA was synthesized from the obtained RNA sample using the BioArray RNA Transcript labeling Kit (Affymetrix, Santa Clara, CA).
Hybridization protocol
Biotin-labeled cRNA was fragmented in a 40-μl reaction mixture containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30mM magnesium acetate, and incubated at 94°C for 35 min, and then hybridized onto the MGU74v2 series of arrays.
Scan protocol
GeneChips were scanned with a GeneArray Scanner (Hewlett-Packard, Santa Clara, CA), controlled by MicroSuite 5.1 software (Affymetrix).
Description
Gene expression data from GFP-positive cells from P8 retina in APB5-treated homozygous Tie2-GFP transgenic mice
Data processing
The dChip software (version 1.3) was used to normalize the CEL files at probe level and compute model-based expression values using the PM only model.
