Sybr green premix pro taq hs qrt pcr kit

The HiPure RNA Mini Kit (Magen, Guangzhou, China) was used to isolate total RNA following the manufacturer’s instructions. Primer pairs for quantitative real-time PCR (qRT-PCR) were designed using Primer3 (Untergasser et al., 2012 (link)) (Supplementary Table S1). First-strand cDNA was synthesized using the Hiscript QRT SuperMix (with gDNA Wiper; Vazyme, Nanjing, China). qRT-PCR was conducted on a LightCycler 480 real-time PCR system (Roche, Basel, Switzerland) using the SYBR ^® Green Premix Pro Taq HS qRT-PCR Kit (Accurate Biotechnology, AG11718, Hunan, China). The PCR protocol was described by (Ou et al., 2022 (link)). The comparative 2-ΔΔCT method was used to calculate relative expression values for each gene (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted from 200 μl of serum using Trizol reagent (Sigma, USA), and then, RNA was reverse transcribed into cDNA with a high-capacity cDNA reverse transcription kit (Takara). Quantitative real-time PCR (qRT-PCR) analyses were performed by SYBR Green premix pro Taq HS qRT-PCR kit (Accurate Biotechnology (Hunan) Co., Ltd) to validate gene expression, and the level of β-Actin served as an internal control. The relative expression of the target gene was calculated and normalized to the expression of the reference gene β-Actin. The primers’ sequences for qRT-PCR are shown in Supplementary Table 1.

Total RNA was extracted from tissue samples using Trizol reagent (Sigma, USA), and then, RNA was reverse transcribed into cDNA with the Evo M-MLV RT Premix (Accurate Biotechnology (Hunan) Co.,Ltd). Quantitative real-time PCR (qRT-PCR) analyses were performed by SYBR Green premix pro Taq HS qRT-PCR kit (Accurate Biotechnology (Hunan) Co.,Ltd) to validate gene expression, and the level of β-Actin served as an internal control. The relative expression was calculated based on the comparative Ct (2^−ΔΔCt) method. The primers’ sequences for qRT-PCR are shown in Table 2. The protein expression levels of 6 prognostic gene signatures in normal liver and HCC tissues were determined using the Human Protein Atlas (HPA).Table 2

The primer sequences for qRT-PCR analysis

Premier	Sequences (5′–3′)
β-Actin-F	CTCCATCCTGGCCTCGCTGT
β-Actin-R	GCTGTCACCTTCACCGTTCC
TNFSF4-F	CCCTGGGACCTTTGCCTATT
TNFSF4-R	GGGGTTGGACCCTTTCCATC
TNFRSF4-F	AAGCCTGGAGTTGACTGTGC
TNFRSF4-R	CCTGTCCTCACAGATTGCGT
TNFRSF11A-F	GTTGCAGCTCAACAAGGACAC
TNFRSF11A-R	CAGAGAAGAACTGCAAACCGC
TNFRSF11B-F	CTGGAACCCCAGAGCGAAAT
TNFRSF11B-R	GCCTCCTCACACAGGGTAAC
TMIGD2-F	AGAACAGAAACCGGATCGCA
TMIGD2-R	GGCTGTTACCTGAGTCCCTT
CD40LG-F	ATGGGAAACAGCTGACCGTT
CD40LG-R	GATTGTTGCCCGCAAGGTTT