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.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.