Tp8000 thermal cycler dice real time system

Genomic DNA was extracted from the primary tumours and noncancerous mucosa using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). TL was measured using a quantitative (Q)-PCR assay kit (ScienCell Research Laboratories, Carlsbad, CA, USA). Genomic DNA (10 ng) was amplified with a TB Green Premix Ex Taq II system (Takara, Tokyo, Japan) using a Takara Thermal Cycler Dice Real Time System TP8000. The data analysis was conducted according to the manufacturer's instructions. For each DNA sample, two consecutive reactions were performed: the first to amplify a single-copy reference (SCR) gene and the second for the telomere sequence. The SCR primer set recognises and amplifies a 100 bp-long region on human chromosome 17 and serves as a reference for data normalisation. The Q-PCR conditions were as follows: 95 °C for 10 min followed by 32 cycles of 95 °C for 20 s, 52 °C for 20 s, and 72 °C for 45 s. All reactions were performed in triplicate. After Q-PCR was performed, we used the instrument's analysis software to analyse the data 12 (link).

Total RNA was isolated using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized using a ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). The mRNA levels of TET1, TET2, TET3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured via Q-RT-PCR using SYBR Premix Ex Taq (Takara, Tokyo, Japan), the Takara Thermal Cycler Dice Real Time System TP8000 (Takara) and the primer sets presented in Supplementary Table 2. The data were analyzed using the ^ΔΔCt method.