Total RNA was extracted from tissue and primary cultured cells (RNeasy micro kits; Qiagen, West Sussex, UK) in accordance with the manufacturer's instructions. In the initial step, RLT buffer (containing β-mercaptoethanol) was added to Eppendorf tubes containing snap-frozen tissues, or they were added directly to PBS-washed culture monolayers. Cultured cell lysates were removed with a cell scraper. Frozen tissue was homogenized with an Eppendorf homogenizer, and both sample sets were then passed through a needle and syringe. The remainder of the protocol was as described by the manufacturer and included a DNAse step. Quality control was maintained with an RNA analyzer (Bioanalyzer 2100; Agilent, West Lothain, UK) and an RNA lab chip (6000 Nano lab chip; Agilent) to ensure that 28S and 18S rRNA bands were clearly evident in total RNA samples. RNA was quantified with a spectrophotometer (ND-1000; NanoDrop, Wilmington, DE). For the 18 samples analyzed, the 260:280 ratio ranged from 1.8 to 2.2 (mean, 2.0). Where possible, total RNA was immediately used for cDNA generation or was briefly stored at –80°C. Generation of cDNA was performed with reverse transcriptase (Superscript II; Invitrogen, Paisley, UK) and random primers (Promega, Southampton, UK), according to standard protocols.