Cfx96 connect

To prepare the sample mixture, 1 μM WT or mutant DNMT3A PWWP–ADD-MTase protein was dissolved in a 20-μL buffer containing 20 mM HEPES (pH 7.2), 5% glycerol, 250 mM NaCl, 5 mM DTT, and 1× GloMelt Dye. The experiment was conducted using a BioRad CFX96 connect real-time PCR detection system, as previously described^{36 (link)}, with the sample plate subjected to stepwise heating from 4 to 95 °C at 0.5 °C per increment. Fluorescence intensity was recorded with the excitation and emission wavelength set to 470 nm and 510 nm, respectively. The experiments were performed in triplicate.

This study was approved by the Animal Care and Ethics Committee of Zhujiang Hospital of Southern Medical University, China (NO. LAEC‐2020–111). Three‐week‐old male wild‐type C57BL/6 mice (n = 9) were obtained and raised in the animal experimental centre of Zhujiang Hospital of Southern Medical University, and the entire experiments were carried out according to the rules and regulations of the centre. Form‐deprivation myopia was induced by the placement of hand‐made caps from 0.2 mL PCR tubes onto a randomly selected eye for 10 days, as described previously.³¹, ³² C57BL/6J mice were euthanized according to the approved experimental protocol. Fresh retinal specimens from both treated and control eyes were separated under a stereomicroscope and snap frozen in liquid nitrogen. Total miRNA was isolated from each sample using E.Z.N.A.^TM miRNA Kit (R6842‐00, Omega, Bio‐Tek, Norcross, GA, USA) following the manufacturer's protocol. cDNA was synthesized using miRNAFirst Strand cDNA Synthesis (Stem‐loop Method NO. B532453, Sangon Biotech, Shanghai, China). qPCR was performed on a Bio‐Rad real‐time PCR instrument (CFX96 Connect, Bio‐Rad, Hercules, CA, USA) using miRNA qPCR Kit (SYBR Green Method NO. B532461, Sangon Biotech, Shanghai, China). The relative miRNA level was normalized to the internal control U6 using the 2^−∆∆CT method. p value < 0.05 was considered statistically significant.

RNA analyses were made as previously described (26 (link)). cDNA quantification for CD69 and IFNγ, (Bio-rad, CA, USA) was performed by using a real-time PCR (CFX96 connect, Bio-rad, CA, USA) and a SYBR Green PCR mix (Bio Rad), and all reactions were run in duplicate. The results are presented as the media of the relative expression units to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin reference genes calculated by the 2^−ΔΔCt equation using the CFX manager 3.1 (Bio Rad). Reactions were performed according to the following thermal profile: initial denaturation (95°C, 15 min) followed by 40 cycles of 15 s at 95°C (denaturation) and 20 s at 60°C (annealing) and 20 s at 72°C (extension). Melting curve analysis was also analyzed for amplicon identification. Ct values of 35 or higher were excluded from the analyses.

TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA, and cDNA synthesis was performed following the protocol of Transcriptase Kit (Thermo, Waltham, MA). And qRT-PCR was conducted on a CFX96 Connect (Bio-Rad, Hercules, CA) using a SYBR Green PCR kit (Vazyme Biotech, Nanjing, China). The mRNA expression levels were calculated by using the 2^−ΔΔCt method. Results are shown as fold-change relative to the normal group or the DMSO group. Primer sequences were listed in Supplementary Data.

SLC5A11 SNP rs11074656 (C/T), DRD2 SNPs rs6276 and rs6277 were identified using Taqman PCR probes (Thermo Fisher), a two-step fast cycling real-time PCR machine (BioRad CFX96 Connect, Hercules, CA, USA), and SNP determination by CFX Maestro 2. SLC5A11 mRNA was quantified using real-time quantitative reverse transcriptase PCR (qRT-PCR), as previous described [77 (link)]. Primers: SLC5A11 sense 5′-GCCTCCACAGTTAGATCCCC-3′; SLC5A11 anti-sense 5′-CAGAACTAGCACCGCGATG-3′; actin sense 5′-AGAAAATCTGGCACCACACC-3′; Actin anti-sense 5′-CTCCTTAATGTCACGCACGA-3′.