Real-time quantitative polymerase chain reaction analysis

Total RNA was extracted from tissue or cells using QIAzol Reagent (Qiagen) according to the manufacturer’s instructions. Reverse transcription of 1 μg of RNA was performed by using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time quantitative polymerase chain reaction (QPCR) was performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Relative amounts of all mRNAs were calculated using the comparative C_T method normalized to the reference gene Rpl13a, 36B4, or Hprt1. The primer sequences (sense and antisense) for mouse are listed in table S2. Absolute quantification of PKD isoform copy numbers in the liver was performed according to the standard protocol of Applied Biosystems. Briefly, the primers were designed to be located within same exons (table S2), and genomic DNA of known concentration was used to create a standard curve reflecting copy numbers.