Sybr green

In brief, total RNA was isolated from specific treatment cells using Trizol reagent). Using Accupower RT Premix (Bioneer), 1 μg of total RNA was converted to cDNA. Quantitative real-time PCR reaction was performed by using the Green Star PCR Master Mix where SYBR Green was included (Bioneer). Then, the specific genes CAT, SOD1, SOD2 and GPx were determined by the Exicycler Real Time Quantitative Thermal Block (Bioneer). The specific primers were previously reported by our research group [19 (link)]. The relative expression of each gene was normalized to the internal control gene (β-actin).

MIHA (Human normal liver cell), LX-2 (Human normal stellate cell), and four human HCC cell lines, including huh7, HepG2, Li-7, and PLCPRF5, were all purchased from ATCC, and were cultured in DMEM (Gibco) supplements with 10% fetal bovine serum (Hyclone), 100 U/ml of penicillin (Gibco), and 0.1 mg/ml of streptomycin (Gibco) at 37°C in a humidified atmosphere of 95% O₂, 5% CO₂.
Total RNA was isolated using the TRIpure Reagent (RP1001, Bioteke). Reverse transcription was performed on 1 mg of RNA at 60°C for 35 min using a BeyoRT II M-MLV (D7160L, Beyotime). After reverse transcription, the cDNAs were used for semi-quantitative PCR by using 2×Taq PCR MasterMix (PC1150, Solarbio) and SYBR Green (SY1020, Solarbio). Amplification was carried out as recommended by the manufacturer in the qPCR instrument (Exicycler 96, BIONEER): 25 µl of reaction mixture contained 12.5 µl of SYBR Green mastermix, the appropriate primer concentration, and 1 µl of cDNA. The amplification program included the initial denaturation step at 95°C for 10 min, 40 cycles of denaturation at 95°C for 10 s, and annealing and extension at 60°C for 1 min. Fluorescence was measured at the end of each extension step. After amplification, melting curves were acquired and used to determine the specificity of the PCR products. 2 ^-△△CT method was used in data process.
The sets of specific primers as follows:

RNAs were extracted from the transfected HUVEC cells with RNeasy Mini Kit (QIAGEN, 74104, CA, USA) following the manufacturer's instructions. Total RNAs were reverse‐transcribed to generate cDNA using the AccuPower^® RocketScript™ RT Premix (Bioneer, K‐2101, Korea). The mRNA expression levels of the target genes were measured using SYBR green (Bioneer, K‐6251, Korea). The cycling parameters were as follows: 95°C for 10 min, followed by 40 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C. The mRNA expression level of each gene was normalized with the value of GAPDH mRNA expression. The sequences of primers (Bioneer, Korea) used for the RT–PCR are in Appendix Table S2.

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The amount and purity of RNA were determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). A total of 1 μg of RNA was converted into cDNA with the amfiRivert Platinum cDNA Synthesis Master Mix (GenDepot). qRT-PCR was performed with a 7300 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) using SYBR Green (Bioneer, Daejeon, Republic of Korea). Relative gene expression was evaluated by the comparative CT method and normalized using β-actin. The following primer sequences were used for amplification: (1) iNOS: sense primer, 5′-TCC TAC ACC ACA CCA AAC-3′; antisense primer, 5′-CTC CAA TCT CTG CCT ATC C-3′; (2) β-actin: sense primer, 5′-CTG ACT ACC TCA TGA AGA TCC TC-3′; antisense primer, 5′-CAT TGC CAA TGG TGA TGA CCT G-3′; (3) IL-6: sense primer, 5′-AGG CTT AAT TAC ACA TGT TCT CTG G-3′; antisense primer, 5′-TTA TAT CCA GTT TGG TAG CAT CCA T-3′; (4) IL-1β: sense primer, 5′-GCC ACC TTT TGA CAG TGA TGA G-3′; antisense primer, 5′-AGT GAT ACT GCC TGC CTG AAG-3′.

Single-stranded cDNA was synthesized from 1 to 2 μg RNA using a Quantitect® Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Real-time RT-PCR was performed in duplicate using a CFX96 real-time RT-PCR system (Bio-Rad, USA) with SYBR Green (Bioneer, Korea). Expression levels of target genes were determined using the primers shown in Table 1. Gene expression levels were quantified using ∆∆C_t [40 (link)]. The β-actin gene was used as the reference to normalize the relative expression levels of individual transcripts. The data were analyzed using the Bio-Rad CFX software.Table 1

Primer sequences of macrophage genes used for qRT-PCR assays

Gene name	Forward primer	Reverse primer
b-actin	5’-CGCCACCAGTTCGCCATGGA-3′	5’-TACAGCCCGGGGAGCATCGT-3
Il1b	5’-CAACCACACAAGTGATATTCTC-3′	5’-GGATCCACACTCTCCAGCTGC-3
Il6	5’-TCCAGTTGCCTTCTTGGGAC-3’	5’-GTACTCCAGAAGACCAGAG-3’
Tnf	5’-CACAGAAAGCATGATCCGCGA-3’	5’-CGGCAGAGAGGAGGTTGACTT-3’
Il10	5’-TGGCCCAGAAATCAAGGAGC-3’	5’-CAGCAGACTCAATACACACT-3’
Ifng	5’-TGAACGCTACACACTGCATCT-3’	5’-CGACTCCTTTTCCGCTTCCTG-3’
Mcp1	5′- GGTCCCTGTCATGCTTCTGGG-3’	5’-TCCAGCCTACTCATTGGGATC-3’
P50	5’-CTCACTCAATATTTAATGCAG-3’	5’-CCCTCCGTGTGATGGGCCTTC-3’
P52	5’-CCCATGGAGGTTTGCCAGGTG-3’	5’-CCCACCAGACTGTGGGCATGC-3’
P65	5’-CATCCACATGAACTTGTGGGG-3’	5’-CTGGCTAATGGCTTGCTCCAG − 3’

Two–three weeks after sorting, MICA/B-negative single-cell clones were established and cultured on 6-well plates (n = 188). Clonal genomic DNA was isolated from 1 × 10⁵–10⁶ cells from each clone using a TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Amplification of each target region was carried out using PCR with forward and reverse primers: MICA, 5′-CGTTCTTGTCCCTTTGCCCGTGTGC-3′ (forward) and 5′-GAATTGGAGGGAGAGGAGAGC-3′ (reverse); MICB, 5′-AGCCCCACAGTCTTCGTTAC-3′ (forward) and 5′-CCAGGGTCGGTACCTGTTCT-3′ (reverse). The PCR program consisted of one cycle of 95 °C for 3 min, 35 cycles of 98 °C for 20 s, 65 °C for 15 s, and 72 °C for 20 s; one cycle of 72 °C for 2 min; and one cycle of 10 °C for 5 min. PCR products were analyzed by Gel Doc XR + system (Bio-Rad, Hercules, CA, USA) on 2% agarose gels, using SYBR Green and a 100-bp ladder (Bioneer, Daejeon, Korea) for screening of predicted large deletion clones. PCR products of selected clones were analyzed by Sanger sequencing (Cosmo Genetech, Seoul, Korea) using the same primers.

The expression level of miR-34a was determined by qRT-PCR. Briefly, total RNA was isolated from cells using the RNApure Total RNA Fast Isolation Kit and was then reverse-transcribed using the Power RT Kit. Real-time PCR was performed using 2x Power Taq PCR MasterMix and SYBR Green on a Superior 5-color Real-Time Quantitative PCR system (Bioneer, Korea). The primers for mature miR-34a and U6 were purchased from Sangon Biotech (China). The primers used are listed in Table 2. The relative amount of miR-34a was normalized to the U6 snRNA, and the fold change in each miRNA was calculated using the 2^-ΔΔCt method [26 (link)].