Abi7300 fluorescence quantitative pcr instrument

Trizol Reagent (Vazyme, NanJing, China) was used to extract total RNA from liver tissue, which was then treated by deoxyribonuclease I to remove the contaminant DNA. RNA was quantified based on the absorption of light by a Nanodrop ND-2000c spectrophotometer (Thermo Scientific, Camden, UK) at 260 nm (A260) and 280 nm. From each sample, 1 μg of RNA was used to synthesize cDNA in a 20 μL reaction mixture using the Primer-ScriptTM reagent kit (TaKaRa, Dalian, China) according to the manufacturers’ instructions. The real-time quantitative polymerase chain reaction was carried out by using the SYBR Premix Ex Taq II kit (TaKaRa) in an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). The 20 μL reaction system included 10 μL of SYBR Premix Ex Taq buffer, 0.4 μL each of forward and reverse primers and dye, 2 μL of cDNA template, and 6.8 μL of distilled water. The real-time PCR cycling conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The relative mRNA expression was determined using β-actin as an internal reference gene. The significance and correlation of quantitative results were analyzed by using 2‒ΔΔct as per Livak and Schmittgen [19 (link)]. Primer sequences are shown in Table 2.

Human normal thyroid cell line Nthy-ori3-1 (derived from human thyroid follicular epithelial normal cells) and human thyroid cancer cell lines (BC-PAP, K1, and TPC-1) were purchased from Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were cultured in Roswell Park Memorial Institute (RPMI, Keygen, Nanjing, China) 1640 complete medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin in an incubator at 37°C with 5% CO₂ and saturated humidity. The CO₂ cell incubator purchased from Forma Scientific UK. ABI7300 fluorescence quantitative PCR instrument was purchased from Applied Biosystems Inc.