RNA isolation and qPCR analysis

RNA was isolated from tissues using a commercially available kit (MicroRNeasy Kit, Qiagen, Hilden, Germany). Identical amounts of RNA were reverse-transcribed to cDNA using Superscript III (Invitrogen, Darmstadt, Germany) and gene expression was analyzed using TaqMan probes (ThermoFisher Scientific Inc., Rockford, IL USA) using a ViiATM7 Real-Time PCR System or QuantStudio 6 FLEX Real-Time PCR System (ThermoFisher Scientific Inc., Rockford, IL USA). Expression changes were calculated using the 2^−ΔΔCt method normalized by Hprt as housekeeping gene. When indicated, qPCR expression analysis was conducted on cDNA derived from immunoprecipitated RNA of OT:RiboTag mice following reverse (SMARTer PCR cDNA synthesis kit; Takara Bio Inc., Shiga, Japan).