Quantification of Zebrafish Liver Gene Expression

Total RNA was extracted from independent pools of micro-dissected zebrafish livers using the RNeasy Micro Kit (QIAGEN, #74004). RNA integrity was assessed by a High Sensitivity RNA ScreenTape assay (Agilent, #5067-5579) on a 2200 TapeStation. cDNA was generated from 1 to 10 µg RNA using the Superscript III First Strand Synthesis System (Invitrogen, #18080051) and oligo(dT) priming according to the manufacturer’s instructions. RT-qPCR was performed using a SensiMix SYBR kit (Bioline, #QT605-05) on an Applied Biosystems ViiATM7 Real-Time PCR machine. Expression data were normalised by reference to hrpt1, b2m, and tbp. LinRegPCR V11.0 was used for baseline correction, PCR efficiency calculation and transcript quantification analysis (Ruijter et al., 2009 (link)). Relative expression levels were calculated by the 2^−∆∆Ct method and all results were expressed as the mean ± standard error of the mean (SEM) of three independent pools of biological replicates. Primer sequences for RT-qPCR are listed in Table 2.