Quantitative RT-PCR analysis of gene expression

Total RNA was extracted from 30 to 40 dissected livers for each condition using the RNeasy Micro Kit (Qiagen, Hilden, Germany); cDNA was synthesized from the RNA using the SuperScript^® III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocols. qRT-PCR was performed as previously described^{14 (link)} using the QuantStudio 12K Flex machine (Applied Biosystems, Waltham, MA) with the iTaqTM Universal SYBR^® Green Supermix (Bio-Rad, Hercules, CA). eef1a1l1 was used for normalization as previously described^{60 (link)}. At least three independent experiments were performed. For each replicate with larval livers, the control value was first set to 1, and then the values of the experimental groups were calculated relative to the control value. The primers used for qRT-PCR are listed in Table S3; the Ct values of qRT-PCR data are listed in Table S4.