RNA was isolated from cells using an RNeasy kit (Qiagen) according to the manufacturer’s protocol. For tissue, animals were anesthetized (350 μg/kg chloral hydrate, i.p.) and the relevant tissue or brain region was rapidly dissected out and snap-frozen in liquid N2 or immersed in RNA later for 12 to 16h at 4°C. Total RNA was isolated from the tissue using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA was then treated with DNase (RNase-Free; Roche) and post-cleaned using an RNeasy kit (Qiagen). 1 μg of total RNA was reverse-transcribed to cDNA using Superscript III (Invitrogen) and random hexamers. cDNAs from samples were amplified and detected using SYBR Green I reagent (Roche) and a LightCycler 480 Instrument (Roche), respectively. Quantification of mRNA levels was performed using the LightCycler 480 Software (Roche) using standard curves and normalizing to β-Actin or L32 expression.
