Rox reference dye 2

BM mononuclear cells (BMMNCs) were extracted by Lymphocyte Separation Medium (TBD sciences, Tianjin, China). Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription (RT) was performed as reported previously 25 (link),26 (link).
Real-time quantitative PCR (RQ-PCR) was performed on a 7500 Thermo cycler (Applied Biosystems, CA, USA). The reaction system with 20 μL volume consisted of H₂O 6 μL, 10 μM of AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Piscataway, NJ, USA), 0.4 μM of ROX Reference Dye 2 (Invitrogen, Carlsbad, CA,USA),and 0.8 μM of primers (forward 5'-ACAGGAGGTTGTCTGCACACG-3' and reverse 5'-GACTTGAGGAACTGCTTCTCCG-3') 20 (link). The RQ-PCR reaction conditions were 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, 65 °C for 30 s, 72 °C for 32 s, and 86 °C for 32 s to collect fluorescence, finally followed by 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s, and 60 °C for 15 s. Both positive and negative controls were included in each assay. Relative NKD2 expression levels were calculated using the following equation:

Genomic DNA was isolated using genomic DNA purification kit (Gentra, Minneapolis, MN, USA) and was modified using the CpGenome DNA Modification Kit (Chemicon, Ternecula, Canada) according to the manufacturer's instructions. The primers used for the methylated (M) [(forward) 5'-GATCGTAGGGGATAGTTTCGTGGC-3'; (reverse) 5'-AAAACAACTCTAACACCGCTCCCCG-3'] and unmethylaed (U) [(forward) 5'-TGGATTGTAGGGGATAGTTTTGTGGT-3'; (reverse)5'-CAAAAACAACTCTAACACCACTCCCCA-3'] 20 (link). Real-time quantitative methyaltion-specific PCR (RQ-MSP) reaction contained 0.8 μM of primers, 10 μM of AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Piscataway, NJ, USA), 0.4 μM of ROX Reference Dye 2 (Invitrogen, Carlsbad, CA, USA), and 20 ng of modified DNA was also performed a 7500 Thermo cycler (Applied Biosystems, CA, USA). The reaction condition was 95°C for 5 min, 40 cycles for 10 s at 95°C, 30 s at 62°C (M) or 64°C (U), 72°C for 30 s, and 80°C (M) or 76°C (U) for 30 s, eventually a melting program of one cycle at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, and 60°C for 15 s. The normalized ratio (N_M-NKD2) was applied to assess the level of NKD2 promoter methylation in samples. N_M-NKD2 was calculated using the following formula: