Robust Quantification of Gene Expression

Total RNA was isolated with RNeasy^® Mini Kit 250 (QIAGEN, Hilden, Germany). The homogenates of individual tissues from at least five animals, or five whole animals, of each genotype were combined together for RNA extraction. The RNA concentration was measured by using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). The RNA from each genotype was reverse-transcribed with the QuantiTect reverse transcription kit (QIAGEN). The cDNA was analyzed by using the SYBR Green-based qPCR method. The PCR primers for the internal control gene rpl8 were described previously [36 (link),37 (link)]. All expression data were normalized against that of the internal control gene rpl8. The expression analyses were performed at least twice, with consistent findings. The primer sequences are listed in Tables S2 and S3.