Tb green premix ex taq 2 system

Total RNA was isolated using TRIzol reagent (Takara, Beijing, China) according to the manufacturer's instructions. One microgram of total RNA was used to reverse transcribe complementary DNA (cDNA) with the TB Green® Premix Ex Taq™ II system (Takara, Beijing, China). Real-time PCR (PrimeScript™ RT Reagent Kit, Takara, Beijing, China) analysis was performed using a StepOnePlus Real-Time PCR System according to the manufacturer's instructions. Primer sequences were as follows: for STMN1, 5′-AAGAGAACCGAGAGGCACAAATGG-3′ (forward), 5′-GGCAAAGGGCAGGAACAGAGTG-3′ (reverse); for E2F1 5′-CTGTGCCCTGAGGAGACCGTAG-3′ (forward), 5′-GAGATGATGGTGGTGGTGACACTATG-3′ (reverse). The data were analyzed by the 2^−ΔΔCt method, and β-actin was used as the reference gene.

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).

Twelve differentially expressed genes were selected for qRT-PCR analysis (Table S5). The Oligo7 (version 7.56) software was used to design primers. PrimerScript RT reagent kit with a gDNA eraser (Perfect Real Time) (TaKaRa, RR047A) was used to synthesize cDNA according to the manufacturer’s protocol. Three technical replicates were performed for each sample. The final reaction volume of 10 µL contained 2 µL of cDNA, 8 µL of qPCR master mix (5 µL of TB Green Premix Ex Taq II system according to the manufacturer’s instructions (TaKaRa, RR820A), 0.25 µL of each primer, and 2.75 µL of RNA-free water). The samples were run on a CFX96 Touch Real-Time System (Bio-Rad, Hercules, CA, USA). The conditions were 94 °C for 30 s, followed by 39 cycles of 94 °C for 5 s and 65 °C for 34 s. The relative mRNA levels of the candidate key genes were calculated using the 2^–ΔΔCt method.