Prostate cancer cell line LNCaP treated for 4 h with R1881.
Extracted molecule
total RNA
Label
Cy3
Hybridization protocol
The probe was heated for 2 min at 95 °C, mixed with 72 µl hybridization buffer no. 1 from Ambion and applied on the cDNA microarray. Hybridization was performed o/n at 45 °C.
Scan protocol
After hybridization, the microarrays were washed and scanned (ScanArray Express HT, PerkinElmer, Boston, MA). Fluorescence intensities on scanned images were quantified using Imagene 5.1 (BioDiscovery, Marina del Rey, CA).
Description
The LNCaP cell line was maintained in RPMI media with 10% fetal calf serum and penicillin/streptomycin. Before R1881 treatment, cells were androgen-deprived by culture for 72 h in medium containing 10% dextran-filtered, charcoal-stripped fetal calf serum with a medium replacement after 36 h. After androgen deprivation, the medium was supplemented with R1881 to a final concentration of 1 nM. R1881 was dissolved in ethanol. The final concentration of ethanol in the media was 0.1%. In the control, ethanol was added to the same final concentration.
Data processing
Normalization was performed using R-routines (Lowess method; http://cmfdb.nki.nl/). All spots with an R-value below 3 or with an average intensity less than 0.4 of the median spot intensity per subarray were considered to be insufficiently reliable and were excluded from the data analysis.
