Metanephric mesenchyme and ureteric buds were dissected from E13.5 rats. At this stage of development, the ureteric buds have already initiated branching morphogenesis producing cleavage of the initial bulbous ureteric bud into a T-shaped tubule. To isolate the metanephric mesenchyme and ureteric bud, embryonic kidneys were dissected and treated with DNase (100 U/ml; Boehringer Mannheim) and trypsin (1 mg/ml; Sigma) for 10 min at 37C in L-15 media. The mesenchyme was freed with minutia pins (Fine Scientific) and the tissues were transferred to L-15 with soybean trypsin inhibitor (1 mg/ml). Cross-contamination between ureteric buds and metanephric mesenchymes was ruled out by visual inspection and staining with dolichos bifloris lectin, which selectively stains the ureteric bud at this stage. In all, approximately 1,200 rat ureteric buds and 240 rat mesenchymes were harvested to prepare RNA. Total RNA from ureteric buds and metanephric mesenchymes was extracted using RNeasy mini kit (Qiagen, Valencia, CA) with on-column DNase digestion according to the manufacturer´s instructions. Targets were produced using standard Affymetrix procedures from 1 ug of total RNA. 2 replicates of the target labeling process were analyzed for each sample. Keywords = Rattus norvegicus, metanephric mesenchyme, ureteric bud, molecular screen for spatially restricted transcripts
Submission date
Nov 13, 2004
Last update date
Oct 28, 2005
Contact name
Kai Martin Schmidt-Ott
Organization name
Max-Delbrueck Center for Molecular Medicine
Department
Differentiation and Regeneration of Kidney Epithelia
