RNA was extracted from the samples using Trizol (Invitrogen) before further purification using the RNeasy MiniElute Cleanup Kit (Qiagen), both according to the manufacturer's protocols.
This protocol describes Agilent's recommended procedures for hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based one-color gene expression analysis.
This protocol describes Agilent's recommended procedures for the preparation and labeling of complex biological targets for Agilent 60-mer oligonucleotide microarray-based one-color gene expression analysis.
array scanning protocol
Arrays were scanned using the Agilent Microarray Scanner Bundle. Raw data were imported and analyzed in J-Express software (http://jexpress.bioinfo.no). Mean spot signals were used as intensity measure and expression data were quantile normalized. The intensities were further log2 transformed. Differentially expressed genes were identified using the Feature subset selection (FSS) method. Only genes that changed more than 3.0 fold with p-values<0.01 were considered as differentially expressed.
EPT2-D5: The prostate cell line EPT2-D5 was grown in MCDB153 medium (Biological Ind. Ltd., Israel) supplemented with 1% MEM non-essential amino acids solution, 200 nM hydrocortisone, 10 nM triiodothyronine, 5 µg/ml insulin, 5 µg/ml transferrin, 5 µg/ml sodium selenite, 100 ng/ml testosterone, 5 ng/mL EGF, 50 µg/mL bovine pituitary extract (Invitrogen), 100 U/ml penicillin, 100 µg/ml streptomycin and 5% fetal calf serum (FCS). Prostate cancer PC3 and DU145 cell lines were grown in Hams F12 medium and DMEM medium (Lonza, Basel, Switzerland) containing 10% FCS, respectively. The medium was changed every 3 days. To achieve expression of lentiviral pGF-STAT3 reporter and pGF-CMV control vector, both vectors were transfected into 293FT cells (Invitrogen, Carlsbad, CA, USA) with Lipofectamine 2000 transfection reagents (Invitrogen). Pseudotyped viral particles were collected 2 days after the transfection of lentiviral plasmids and used for transduction of EPT2-D5 or EPT3-M1 cells. D5HS: To generate D5HS, EPT2-D5 cells were seeded in T75 tissue culture flasks (TPP, Trasadingen, Switzerland) to reach 30% confluence. On the second day, the complete medium was removed and cells were washed twice with PBS and cultured in basic Hams F12 medium (Lonza, Basel, Switzerland) without serum (protein free medium). The medium was changed every 3 days. When the culture reached confluence, cells were trypsinized and the reaction was quenched by soybean trypsin inhibitor (Invitrogen), the cells were pelleted, resuspended and split to 2 new flasks. The protein free medium was changed every 3 days until the spheroids were generated about 2 weeks later. To passage the prostate spheroids, culture medium was collected in 15 ml centrifuge tubes and kept at room temperature for 10 minutes to let spheres settle down to the bottom of tubes. The upper 9/10 of medium was gently removed and the remaining lower part contained most of the big spheres (tight, spherical, >100μm in diameter) and was gently centrifuged at 800 rpm for 2 minutes. The pelleted spheroids were dissociated enzymatically (0.05% trypsin, 0.53 mM EDTA) and mechanically (pipetting). The dissociated cells were analyzed microscopically for single-cellularity. The single cells were transferred to and attached in new T75 tissue culture flask. The protein medium was changed every 3 days until new spheres again were generated after around 10 days. EPT3 (TC-3), EPT3-PT1, EPT3-M1: We tested the tumorigenicity of D5HS by subcutaneous injection of the spheres in BALB/c nude mice. Cells recovered from the subcutaneous tumors were named EPT3 (TC-3). To obtain metastatic tumors, luciferase expressing EPT3 cells were inoculated orthotopically into the prostate of mice. Large primary tumors and extensive abdominal metastasis were found in 6 weeks (Figure 2C). Cells recovered from the primary tumor and the metastases were named EPT3-PT1 and EPT3-M1, respectively.