RNA and Protein Isolation from Thyroid Tissue

RNA and protein were isolated from thyroid tissue samples and cells with the Tri reagent system (Sigma-Aldrich) according to the manufacturer's instructions. A few random RNA samples were run on the Agilent Bioanalyzer 2100 to ensure adequate quality. For the tissue samples, 20 µg of RNA were further purified with the RNEasy micro kit (Qiagen, Hilden, Germany) according to the kit instructions to remove residual PCR inhibitors. Reverse transcription was performed with the iScript cDNA synthesis kit (Bio-Rad Life Science, Hercules, CA, USA). RT-qPCR reactions were run on a CFX96 system (Bio-Rad Life Science) using the iTaq Universal SYBR Green Supermix kit (Bio-Rad Life Science), as previously described.²² (link)
HPRT1, ACTB and SDHA were chosen as reference genes for the tissue samples, based on a previous report,²³ (link) whereas HPRT1 and ACTB were used for the cells. Primer sequence and efficiency information can be found in Supplementary Table S2.