cell line: LNCaP cell type: androgen-sensitive human prostate adenocarcinoma
Growth protocol
LNCaP cells were grown at 37ºC with 5% CO2 in T medium with 10% fetal calf serum. PrEC cells were grown at 37ºC with 5% CO2 in PREGM media (Cambrex).
Extracted molecule
total RNA
Extraction protocol
Total input RNA was isolated as per the manufacturers instructions (Millipore, Cat# 17-700). Briefly, Cells were harvested at 80-90% confluency. After two ice-cold PBS washes, cells were scraped and collected by centrifugation at 1500 rpm for 5 minutes at 4C. The pellet was resuspended in an equal volume of lysis buffer and incubated on ice for 5 mins before being frozen at -80 C to complete the lysis process. The lysate was thawed quickly and centrifuged at 14,000 rpm for 10 mins at 4C. 100ul of the supernatant was added to 900ul of buffer. 10 ul was removed as input RNA. The RNA was diluted with 107 ul wash buffer, 15ul of 10% SDS, and 18 ul proteinase K to a total volume of 150 ul. After incubation at 55 C for 30 mins with shaking to digest the protein, centrifuge briefly, and transfer supernatant into new tube. 250 ul wash buffer was added to each tube. Phenol:chloroform:isoamyl alcohol extraction was performed with an equal volume, followed by chloroform extraction, and the RNA was precipitated by adding 50 ul of Salt Solution I, 15 ul of Salt Solution II, 5 ul of Precipitate Enhancer and 850 ul of absolute ethanol and stored at -80C overnight. After centrifugation at 14,000 rpm for 30 mins at 4C, the pellet was washed once with 80% ethanol, before being re-suspended in 10-20 ul RNase-free water. RNA quality was assessed by RIN using a RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100 (Agilent Technologies).
Label
biotin
Label protocol
Total RNA was fragmented and biotin labeled at the Ramaciotti Centre for Gene Function Analysis at the University of New South Wales, Kensington, NSW, Australia), according to the manufacturers instructions.
Hybridization protocol
Samples were hybridised on Affymetrix HuGene 2.0ST arrays at the Ramaciotti Centre for Gene Function Analysis at the University of New South Wales, Kensington, NSW, Australia), according to the manufacturers instructions.
Scan protocol
GeneChips were scanned using a GC3000 scanner at the Ramaciotti Centre for Gene Function Analysis at the University of New South Wales, Kensington, NSW, Australia), according to the manufacturers instructions.
Description
Gene expression data from LNCaP cell line. LNCaP_rep2
Data processing
Affymetrix CEL files were RMA normalised using the rma function as implemented in the R package oligo version (1.28.2).
