cell line: LNCaP (CRL-1740) tissue: prostate cancer cells metastasized to a lymph nodes
Total RNAs were isolated from the prostate cancer cell lines using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) and treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Reverse transcription of RNA was carried out in a final volume of 20 mL containing buffer, 0.1 M DTT, Oligo (DT)20, 10 mM dNTPs, 0.4 U/mL of RNase inhibitor, 1.25 U/mL of reverse transcriptase (Invitrogen Corp) and 1 mg of total RNA.
Cyanine-3 (Cy3) labeled cRNA was prepared from 100ｎg RNA using the Low Input Quick Amp labeling kit, one-color (Agilent Technologies) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
480ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 25 x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE Microarrays (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent Technologies) and 1 minute with 37°C GE Wash buffer 2 (Agilent Technologies), then dried immediately.
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slide (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
Gene expression in TSA treated prostate cancer cell line
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent Technologies) using default parameters (protocol GE１_1010_Sep10 and Grid: 028004_D_F_20110325). Genes-screening was performed using the GeneSpring GX v12.0.0 software package (Agilent Technologies).