A comparison of forskolin-induced mRNA vs. an inactive control in the rat PC12 cell pheochromocytoma cell line.

PC12 cells (passage 22) were cultured at 5% CO2 in a 37 C incubator. The growth medium was DMEM (high glucose, Invitrogen) supplemented with 25mM HEPES, 10% FCS, 5% FBS and 1x Pen/Strep (Invitrogen). 1x106 cells were starved overnight in DMEM+25 mM HEPES and treated with 10 uM 1,9 dideoxy forskolin or forskolin for 60 min. The final DMSO concentration was 0.05%. Total RNA was purified using Trizol (Invitrogen) according to the manufacturers protocol. An additional round of purification was conducted using Rneasy columns (Qiagen) according to the manufacturers protocol. cRNA was synthesized and labeled according to standard Affymetrix protocols. 2 biological replicates for the forskolin and 1,9 dideoxy forskolin conditions were run on Affymetrix RAE230 A and B chips for a total of 8 hybridizations. Keywords: repeat sample