Trizol reagent lysis buffer

Total RNA was extracted from tissues using the TRIzol reagent lysis buffer (Invitrogen, Carlsbad, CA) from fresh snap-frozen samples according to the manufacturer's protocol. Real-time PCR (qPCR) was performed in an ABI PRISM 7900HT Sequence Detector (Applied Biosystems, Milan, Italy) using the relative quantification method (2^−ΔΔCt method) as previously described [17 (link)]. Primer sequences for PTTG1 and AURKA were derived from previous works [18 (link), 19 (link)]. Data were analyzed with the Sequence Detection Software rel. 2.4 (Applied Biosystems), adopting an automatically set baseline and a fluorescence threshold adjusted to measure quantification cycle (Ct) values. Validation experiments performed using the standard curve method with five serial dilutions of genomic DNA from control subjects showed identical amplification efficiencies (100%±10%) calculated according to the formula E = 101/−slope − 1 for all assays. Using the 2^−ΔΔCt method, the data were presented as the fold change in gene expression normalized by a reference gene and relative to a calibrator sample. As the reference gene in this study, we used β-actin, one of the most commonly used housekeeping genes. A pool of cDNA derived from mixed normal human thyroid tissues was used as the calibrator source in our study.

Each surgical specimen was snap-frozen in liquid nitrogen within 15 minutes of collection and stored at -80°C pending RNA recovery. Total RNA was extracted using the TRIzol reagent lysis buffer (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer's protocol.