|Public on Dec 31, 2009
|Vegetal cortex rep2
|stage VI oocyte
|developmental stage: full-grown, stage VI oocyte
isolation: collagenase treatment
treatment: manual dissection of vegetal cortex
cell part: vegetal cortex
|Manual isolation of vegetal cortices was performed using the methods of Elinson et al. (1993), with modifications. Briefly, collagenased stage VI oocytes were transferred to 1x MEM (100 mM MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO4,) in a clean glass dish and bisected into animal and vegetal halves using a new scalpel blade. The vegetal halves were placed cortex side down onto a nitrocellulose filter, and a cover slip was used to compress away the yolky cytoplasm. After the cover slips were removed, gentle agitation was used to free the cortex from the membrane and to dislodge any additional yolk. Isolated cortices were rinsed in fresh MEM and frozen in batches of ten on dry ice. Intact stage VI oocytes from the same female were frozen as controls.
|Total RNA was prepared by proteinase K digestion followed by phenol extraction and ethanol precipitation. Purified RNA was treated with DNase prior to cDNA synthesis. RNA quality was verified using the Experion System (BioRad) for automated electrophoresis.
|100 ng of total RNA was used to make complementary RNA (cRNA)d, which was amplified using the GeneChip® Two-Cycle cDNA Synthesis Kit (Invitrogen/Affymetrix). cRNA labeling, hybridization and data acquisition were performed by The University of Iowa, Carver College of Medicine DNA Core Facility, using standard Affymetrix protocols and reagents.
|Labelled cRNA was hybridized to Xenopus laevis Genome Arrays (Affymetrix) at 45˚C using Model 640 hyb ovens, processed using the Model 450 GeneChip fluidics station (Affymetrix).
|Image files were captured on a Model 3000 GeneArray scanner and processed using Affymetrix micorarray analysis software.
|Sample Number: 3
Replicate Number: 1
Experimental condition: Experimental sample
|Quality control included standard Affymetrix internal controls. GeneChip Operating software was used to generate data files (CEL), and MAS 5 for calculating expression signals.
|Aug 18, 2009
|Last update date
|Aug 19, 2009
|Douglas W. Houston
|The University of Iowa
|Department of Biology
|Microarray analysis of mRNAs enriched in the vegetal cortex of Xenopus oocytes