Real-time PCR

Total RNA from cells and mouse thyroid tissue was extracted by using Qiazol reagent (Qiagen, cat. no. 79306) according to the manufacturer’s protocol. Total RNA from formalin-fixed paraffin-embedded human thyroid cancer tissue was extracted using a High Pure FFPET RNA isolation kit (Roche, cat. no. 06868517001) according to the manufacturer’s protocol. Reverse-transcription was performed with Superscript II (Invitrogen, cat. no. 18064–014) following the manufacturer’s protocol. TaqMan real-time primer/probe sets were purchased from Applied Biosystems: Slc7a5 Mm00441516_m1, SLC7A5 Hs00185826_m1, Slc3a2 Mm00500521_m1, Slc1a5 Mm00436603_m1, Slc38a2 Mm00549967_m1, Atf5 Mm04179654_m1, actb 4352341E, Tuba1a Mm00846967_g1, ACTB 4326315E. Quantifications were performed using a ViiA 7 real-time PCR machine (Applied Biosystems). Relative differences in gene expression levels were assessed by normalizing the Ct value of the target gene to the Ct value of the housekeeping gene and quantified by the 2^-∆∆Ct method.