Cfx96 fluorescence quantitative pcr instrument

Total RNA was extracted from leukemia cells using the HiPure Total RNA Plus Mini Kit (R4121‐02, Magen). Using total RNA as a template, reverse transcription was conducted and cDNA was synthesized with TransScript All‐in‐one first‐strand cDNA Synthesis SuperMix for qPCR (AT341‐01, TransGen). According to the instructions of ChamQ Universal SYBR qPCR Master Mix (Q311‐03, Vazyme), qPCR was performed using a Bio‐RAD CFX96 fluorescence quantitative PCR instrument and the relative expression of the interested genes was calculated by the 2ˆ(ΔΔCt) method. Primers were designed according to the PrimerBank website (https://pga.mgh.harvard.edu/primerbank/). qPCR primers are shown in Table S4 of the Supporting Information.

A TRIzol reagent was used to extract the total RNA of liver tissues (Beyotime Biotechnology Co.). Next, according to the real‐time reverse transcription polymerase chain reaction (RT‐PCR) kit instructions (Beijing ComWin Biotech Co.), and based on the RNA template, the reverse transcriptase enzyme provided was used to reversely transcribe the total RNA into complementary DNA (cDNA). The cDNA was amplified on a CFX96 fluorescence quantitative PCR instrument (Bio‐Rad, Hercules, CA, USA) according to the reaction conditions of the detection kit (Cwbio Century Biotechnology Co.). In brief, samples were treated under the conditions of 95℃ for 10 minutes, 95℃ for 15 seconds (s) and 60℃ for 1 minutes, and the cycle was performed 40 times. Expressions of target genes were carried out by a comparative method (2^−ΔΔCt), with β‐actin used as an internal reference. The primer sequences utilized are shown in Table 1 (Huada Gene Research Institute, Shenzhen, China).

Plasmid pCDNA3.1 (+)-CDbox with known copy number was used as a standard and diluted to 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², and 10 copies/μL according to the gradient of plasmid concentration, and used as a DNA template for the standard curve to perform qPCR amplification using a CFX96 fluorescence quantitative PCR instrument (Bio-Rad). The gene copy number was calculated by the formula: $\mathit{C}\mathit{o}\mathit{p}\mathit{i}\mathit{e}\mathit{s}/\mathit{\mu }\mathit{L}=\frac{\mathbf{6}.\mathbf{02}\times {\mathbf{10}}^{\mathbf{23}}\times \mathit{C}\times {\mathbf{10}}^{-\mathbf{9}}}{\mathit{L}\mathit{e}\mathit{n}\mathit{g}\mathit{t}\mathit{h}\times \mathbf{660}}$ where C is the concentration of the plasmid (ng/μL) and Length is the size of the plasmid sequence (bp).
In this experiment, the upstream primer is 5′-CCC​ACA​ACG​AGG​ACT​ACA​CAC​C-3′ and the downstream primer is 5′-GGG​CTT​GTA​CTC​GGG​TCA​TTG-3'. The reaction system was 2 × qPCR SYBR Green Master Mix (Yeasen) 10 μL, upstream and downstream primers (5 μM) 1 μL each, template 1 μL, add H₂O to make up to 20 μL; amplification conditions for 95°C 5 min; 40 ×: 95°C 10 s, 60°C 30 s. The standard curve was plotted with the log copy number value of the standards as the horizontal coordinate and the measured Ct value as the vertical coordinate, and the amplification efficiency was calculated according to the standard curve. The genome of the cells (2.6 GB) was extracted, and the absolute copy number of the target gene in the sample was calculated by bringing in the Ct value of the cell genome sample.