Biopsied tissue was immediately placed in cold media (Waymouth’s Media 752/1; Sigma, St Louis, MO; with 50 µg gentamicin per ml, Invitrogen, Carlsbad, CA; and 100 U penicillin – 100 µg streptomycin per ml, Sigma) and transported to the lab. Biopsied tissue was washed three times in phosphate-buffered-saline (PBS) containing; 100 U penicillin – 100 µg streptomycin per ml (Sigma, St Louis, MO), and 1.5 µg Amphotericin-B per ml (Invitrogen) and cut into 0.5 cm3 explants. Explants were placed on siliconized lens paper, and cultured in a humidified chamber (5% CO2, 50% O2) at 37C for 2 h in Waymouth’s media supplemented with 5 mg/ml Insulin (Sigma), 0.1 mg/ml Hydrocortisone (Sigma) and 50 μM 5-bromo 2-deoxyuridine (BrdU; COMPANY). The tissue was embedded in Tissue Tek OCT cryostat embedding compound (Ames Co, Division of Miles Laboratory, Elkhart, ID), snap frozen in liquid nitrogen and stored at -80°C for laser capture microdissection.
Extracted molecule
total RNA
Extraction protocol
Laser capture microdissection (LCM) and isolation of total RNA.Serial tissue sections (7 mm) were prepared from OCT preserved tissue with a cryostat microtome using RNase-free techniques and stored at -80°C until use (less than 8 weeks). On day of LCM, tissue sections were fixed and stained in In Situ Hybridization Pap Jars (Evergreen Scientific, Los Angeles, CA) using the Histogene Kit (Molecular Devices, Sunnyvale, CA) following manufacturer’s directions and left in xylene until LCM to ensure tissue was dehydrated. Populations of epithelial cells and intralobular stromal tissue were isolated by LCM using an Arcturus PixCell IIe LCM system (Arcturus Engineering now Molecular Devices) with CapSure LCM Transfer Film (Molecular Devices) according to manufacturer’s protocol. Tissue captured onto transfer film was immediately immersed in 50 ml of isolation buffer from Acturus’ Picopure kit (Molecular Devices), and total RNA was isolated and purified by pooling lysates from several slides of the same samples following manufacturer’s protocol for laser captured frozen tissue. Recovered RNA was subsequently analyzed using a Bioanalyzer 2100 (Agilent Inc; Palo Alto, CA) to determine quality and quantity by use of the Picochip and multiple standards.
Label
biotin
Label protocol
Target preparation was accomplished using NuGen WT Ovation Pico Technology following manufacturer protocol to generate products for the Bovine Affymetrix GeneChip.
Hybridization protocol
Gene chips were hybridized in an Affymetrix 640 Hybridization oven at 45°C for 16 hours with 60rpm rotation. After hybridization, gene chips were washed according to Affymetrix protocol on a Fluidics station using non-stringent (6xSSPE, 0.01% Tween 20, 0.005% antifoam) and stringent (100mM MES, 0.1M NaCl, 0.01% Tween20) wash buffers. Arrays were stained using SAPE stain and antibody solutions. SAPE contains 2ug/ul BSA, 10ug/ml streptavidin Phycoerythrin (SAPE) in 100mM MES, 1M NaCl, 0.05% Tween 20 and 0.005% antifoam. The antibody solution contains: 2mg/ml BSA, 0.1mg/ml goat IgG , 3ug/ml biotinylated anti-streptavidin antibodies in 100mB MES, 1M NaCl, 0.05% Tween 20 and 0.005% antifoam.
Scan protocol
Genechips were then scanned using an Affymetrix Genearray scanner GSC3000, with 7G upgrade. The efficiency of amplification and hybridization were assessed by incorporating Affymetrix Poly-A RNA and Hybridization controls with every sample.
Description
none
Data processing
Gene expression analysis was performed using BioConductor version 2.0 software. Data preprocessing was performed using the RMA method as implemented in the BioConductor package and using updated probe set mappings such that a single probe set describes each gene. Differential expression analysis was tested using moderated t-tests from gene-specific linear mixed model analysis as implemented in the MAANOVA package.
