Sybr premix ex taqtm

From January 2018 to January 2019, 10 pairs of liver cancer tissues and adjacent normal liver tissues were collected from 10 patients who underwent surgical resection and pathological confirmation in the First Affiliated Hospital of Zhengzhou University. The Medical Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University approved this study. Total RNA was extracted by TRIzol reagent (TaKaRa), and cDNA was synthesized by reverse transcription using PrimeScript™ RT Master Mix (TaKaRa) according to the manufacturer's instructions. The levels of GAPDH, CTD-2510F5.4, and DSTNP2 were detected by qRT-PCR using SYBR@ Premix Ex TaqTM (Roche). The results were normalized to the expression of GAPDH. The primer sequences were synthesized by Servicebio (Wuhan, China). GAPDH-F: 5′-GGAAGCTTGTCATCAATGGAAATC-3′, GAPDH-R: 5′-TGATGACCCTTTTGGCTCCC-3′, CTD-2510F5.4-F: 5′-CACCATGCCTGGGTAATTTTAA-3′, CTD-2510F5.4-R: 5′-AGTTCCCTGTTGTCACTGACCTAT-3′, DSTNP2-F: 5′-TGGGCGAAGATGACCTGTTG-3′, DSTNP2-R: 5′-CTTGATTTCTTCTGGTGTGGAGC-3′. The relative expression levels of CTD-2510F5.4 and DSTNP2 were quantitatively calculated by the 2(−ΔΔCT) method.

RT-qPCR was performed to quantify the relative abundance of mRNA for various genes in the YSM samples. Total RNA was extracted from the TRIzol lysate (Invitrogen, Carlsbad, CA, USA), and RNA concentration was determined using a NanoDrop ND-1000 instrument (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA (1 mg) was created from DNA-free RNA using a ReverTra Ace qPCR RT Kit, according to the manufacturer’s protocol (Toyobo, Osaka, Japan).
RT-qPCR was performed with a total volume of 20 μL (10 μL of 2× SYBR Premix ExTaq, 1 μL cDNA mix, 0.5 μL of each primer, and 8 μL of sterile distilled H₂O). An ABI PRISM-7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) was used to detect the amplified products. The primers were designed using Primer Premier 5.0 (Premier, Vancouver, Canada), and their sequences are listed in Table 1. RT-qPCR was performed using SYBR Premix Ex TaqTM (Roche, Basel, Switzerland). All RT-qPCR assays were performed in triplicate. The relative expression of target genes was determined using the 2^−ΔΔCT method [24 (link)] and was normalized to the expression levels of the β-actin internal housekeeping gene.

Total RNA was extracted by TRIzol reagent (TaKaRa), and cDNA was synthesized by reverse transcription using PrimeScript™ RT Master Mix (TaKaRa) according to the manufacturer's instructions. The levels of GAPDH, DLC1 and EZH2 were detected by qRT‐PCR using SYBR@ Premix Ex TaqTM (Roche). The results were normalized to the expression of GAPDH. The primer sequences were synthesized by Sangon (Table 1). The relative expression levels of EZH2 and DLC1 were quantitatively calculated by the 2^(−ΔΔCT) method.

Total RNA was extracted from HCC tissues or xenografts of nude mice or cultured cells according to the manufacturer’s instructions for TRIzol reagent. Total RNA (500 ng) was used for reverse transcription using PrimeScript RT reagent Kit and subjected to quantitative real-time PCR analysis (quantitative PCR, qPCR) using SYBR Premix Ex Taq TM and a Roche’s capillary-based Light Cycler 2.0 Systems. Target mRNA was determined using the comparative cycle threshold method of relative quantification. The calibrator sample was selected from adjacent non-tumor tissues or CON-HepG2 cell samples and β-actin was used as an internal control.

Cells were lysed using Trizol reagent (catalog number: 10296010, Invitrogen, ThermoFisher, USA) to extract total cellular RNA. RNA quality and concentration were determined using UV–visible spectrophotometry (ND-1000, Nanodrop, USA).
To detect mRNA expression, PrimeScript™ RT-qPCR Kit (Cat. No.: RR086A, TaKaRa, Mountain View, CA, USA) was used for reverse transcription. In addition, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed on a LightCycler 480 system (Roche Diagnostics, Pleasanton, CA, USA) using SYBR Premix Ex TaqTM (Cat. No. DRR820A, TaKaRa). Using GAPDH as the internal reference for mRNA analysis. The primers used for amplification were designed and provided by Shanghai General Biosciences Co., Ltd. The primer sequences are shown in Additional file 1: Tables S1 and S2. 2-ΔΔCt represents the relative expression ratio of the target gene in the experimental group compared to the control group, and the formula is as follows: ΔΔCT = ΔCt experimental group—ΔCt control group, where ΔCt = target gene Ct—reference gene Ct [32 (link)].