All in one first strand cdna synthesis supermix for qpcr

High fidelity restriction endonucleases BamHI-HF, HindIII-HF, EcoRI-HF, XbaI-HF, and T4 ligase enzyme were purchased from NEB (New England Biolabs, MA, USA). Bacteria strain DH10B, BL21(DE3), Taq DNA polymerase, and All-in-One First-Strand cDNA Synthesis SuperMix for qPCR were purchased from Transgen, and JM109(DE3) was purchased from Promega (Promega, WI, USA). pT7Oi and pT7Om vectors were already constructed by our group. psilence-2.1-U6-Hygro vector was purchased from Beijing Rambolide Trading Co. DNA fragments were purified with AxyPrep DNA Gel Extraction Kit (Axygen) or DNA Clean-up Kit (Cwbio), and plasmids were extracted by FastPure Plasmid Mini Kit (Vazyme) or Endo-Free Plasmid Mini Kit I (Omega). Cell RNA Kit, BCA protein quantification kit, and Hieff Trans liposomal transfection reagent were purchased from Yeasen. ProtLytic Protein Lysis and Sample Loading was purchased from New Cell & Molecular biotech Co. Human embryonic kidney 293T (HEK293T) was a gift from Professor Yao Shaohua’s laboratory. Michigan Cancer Foundation-7 (MCF-7) and human hepatocellular carcinomas (Hep G2) were purchased from ATCC (American Type Culture Collection, VA, USA).

RNA extraction and qRT-PCR were performed according to previously published methods (Zhao et al., 2022 (link), 2023b (link)). Briefly, the total RNA of the samples was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA) in accordance with the manufacturer's protocol. The RNA concentration and quality were analyzed using a Thermo Scientific NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and all samples had optical density (OD) values between 1.9 and 2.1. The reverse transcription (cDNA) was synthesized from 1 μg of total RNA with All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (AT341-01; TransGen Biotech, Beijing, China). Quantitative Real-time PCR was performed using Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) using 1 μL first-strand cDNA in a volume of 10 μL, and was run in the Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo Scientific, Wilmington, MA, USA). The conditions were as follows: initial denaturation at 94 °C for 30 s, 30 cycles of amplification (denaturation at 94 °C for 5 s, annealing at 56 °C for 15 s, and extension at 72 °C for 10 s), and extension at 72 °C for 10 min. All samples were run in triplicate. β-Actin was used as an endogenous control. The sequence of primers is listed in Table S2. Eventually, the 2^−ΔΔCt method was used to analyze the relative fold changes.

The total RNA from cornea samples was extracted using TransZol Up Plus RNA Kit (ER501-01; Transgen Biotech, Beijing, China) according to manufacturer instructions. Complementary DNA was synthesized using the All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (AE341-03; Transgen Biotech). RT-PCR was carried out using ChamQ SYBR qPCR Master Mix (Q341-02; Vazyme Biotech, Nanjing, China) and the 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Pre-denaturation at 95°C for three minutes was followed by 40 two-step cycles consisting of denaturation at 95°C for five seconds and annealing/extension at 60°C for 30 seconds. Data analysis was conducted using sequence detection system software (Applied Biosystems), with glyceraldehyde-3-phosphate dehydrogenase serving as an internal control for mouse corneas (Table).