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:
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:
