title: S-Adenosylmethionine treatment diminishes the proliferation of castration- resistant prostate cancer cells by modulating the expression of miRNAs
design: S-Adenosylmethionine treatment diminishes the proliferation of castration- resistant prostate cancer cells by modulating the expression of miRNAs
library_construction_protocol: PC-3 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 1.2% penicillin/streptomycin as described previously [10] (PAN-Biotech GmbH, Aidenbach, Germany). For RNA analysis, cells were grown to a subconfluence of 70-80%. The cells were treated with vehicle (0.005 M H2SO4) or 200 μmol of S-adenosylmethionine (SAM) (New England Biolabs, Ipswich, MA) for 120 hours. Under both conditions, the medium was changed every other day.   Total RNA was isolated from PC-3 cells using a NucleoSpin miRNA Kit (Macherey Nagel) according to the manufacturer's instructions.   Following ligation of the RNA to modified 3 ́ and 5 ́ adaptors, the products were reverse transcribed (Super Script III; Thermo Fisher Scientific, Waltham, MA, USA), purified (SPRIselect reagent kit, Beckman Coulter)  and PCR amplified for 14 cycles (KAPA HiFi HotStart PCR Kit, KAPA Biosystems).Size selection was performed by polyacrylamide gel electrophoresis.