Whole embryos were harvested from timed pregnant females at embryonic day 15 (E15). Embryonic bladders were micro-dissected, snap frozen in liquid nitrogen, and stored at -80C. Bladder tissues were homogenized using a Kontes-pellet pestle (Fisher). High quality total cellular RNA was isolated using the Absolutely RNA Miniprep kit according to the manufacture’s protocol (Stratagene, La Jolla, CA). Sample concentration and purity was determined by optical density 260/280 nm ratios using standard procedures. RNA samples were stored at -80C until further use. Total cellular RNA was extracted from whole embryos by homogenizing samples in TRIzol Reagent as outlined by the supplier (Invitrogen, Carlsbad, CA).
Label
biotin
Label protocol
standard Affymetrix protocol
Hybridization protocol
For expression profiling, 25 ng total RNA per sample was processed using isothermal amplification SPIA Biotin System (NuGEN Technologies, Inc.) and 2.2 µg of cDNA was hybridized per microarray. The microarrays utilized were both obtained from Affymetrix (Santa Clara, CA) and included either the Mouse Genome 430 2.0 GeneChip or the Custom GeneChip Chr11/16. After 16 hours of hybridization at 45ºC, washing and staining of microarrays was performed using a Fluidics Station 450 (Affymetrix, Santa Clara, CA). GeneChips were scanned in a GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA). All steps of sample and microarray processing were performed according to manufacturer's recommendations (Affymetrix, Santa Clara, CA).
Scan protocol
standard Affymetrix protocol
Description
n/a
Data processing
Probe set expression estimates were calculated using WPP algorithm (Auer et al, 2007).
