RNA-isolation and Quantitative RT PCR
Total RNA from Pkd2+/+ and Pkd2−/− MEFs was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. 5 μg of total RNA was used for cDNA synthesis with 1:1 mixture of 50 ng/μl random hexamers (Invitrogen) and 0.5 μg/μl Oligo dT (Invitrogen). Quantitative real-time PCR were performed on the resultant cDNA using the PerfeCTa CYBR Green SuperMix for iQ (Quanta BioSciences, Gaithersburg, MD) following manufacturer's instructions. Primer sequence information is presented in Suppl. Table 2. Real-time PCR was carried out using a Bio-Rad iCycler Thermal Cycler (Bio-Rad, Hercules, CA). Reactions were run in triplicate in three independent experiments. The mRNA levels of each target protein were normalized relative to housekeeping gene GAPDH mRNA levels in each sample.