bladder harvested under halothane anesthesia; muscle layer dissected and snap-frozen in liquid nitrogen.
Growth protocol
No specific protocol. Normal animal keeping under DLAR protocol.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Trizol reagent, and purified with Qiagen RNeasy kit
Label
biotin
Label protocol
The purified cDNA were incubated at 37 degC for 4 h in an in vitro transcription reaction to produce cRNA labeled with biotin using MEGAscriptTM system (Ambion, Inc., Austin, TX).
Hybridization protocol
Fifteen to twenty micrograms of cRNA were fragmented by incubating in a buffer containing 200mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc at 958C for 35 min. The fragmented cRNAs were then hybridized with a pre-equilibrated Affymetrix chip at 458C for 14–16 h. After the hybridization cocktails were removed, the chips were then washed in a fluidic station with low-stringency buffer (6x SSPE, 0.01% Tween-20, 0.005% antifoam) for 10 cycles (2 mixes/cycle) and stringent buffer (100 mM MES, 0.1 M NaCl, and 0.01% Tween-20) for 4 cycles (15 mixes/cycle), and stained with SAPE (Strepto-avidin Phycoerythrin). This was followed by incubation with biotinylated mouse anti-avidin antibody, and restained with SAPE.
Scan protocol
Affymetrix HP ChipScanner (G-7 scanner). Software: GCOS1.4
Description
ratbladder_normalcontrol
Data processing
GCOS1.4, normalized through scaled target intensity.
