RNA was extracted from PTC tissues using TRIzol (Sangon Biotech, Shanghai, China). RNA of the thyroid nodule FNA wash-out fluid samples was extracted following the RNA kit protocol (Qiagen Hilden, Germany). Total RNA was first reverse-transcribed into cDNA using PrimeScript™ RT Master Mix (TaKaRa Bio Inc., Japan) (37°C for 15 min, 85°C for 5 s, and cooled to 4°C). RT-PCR of PC and the reference gene β-actin was performed following the protocol for TB Green® Premix Ex Taq™ II (TaKaRa Bio Inc., Japan) in an Applied Biosystems 7500 Real-Time PCR System. The temperature cycling protocol consisted of 30 sec denaturation at 95°C, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. The 40 cycles were followed by 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The PC primers used in this study were 5’-ATGTTGCCCACAACTTCAGCAAGC-3’ (forward primer) and 5’-AGTTGAGGGAGTCAAACACACGGA-3’ (reverse primer). The TGF-βR1 primers used in this study were 5’-GTGACAGATGGGCTCTGCTT-3’ (forward primer) and 5’-AGGGCCAGTAGTTGGAAGTT-3’ (reverse primer). The β-actin primers were 5’-GCACCACACCTTCTACAATG-3’ (forward primer) and 5’-TGCTTGCTGATCCACATCTG-3’ (reverse primer). The PC mRNA expression level was normalized to that of β-actin. Cycle threshold (Ct) values below 35 were used in this study. The 2−Δ Ct of PC mRNA–β-actin mRNA was used to evaluate the expression levels of PC.

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