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2 protocols using cell rna kit

1

Molecular Cloning and Cell Culture Protocol

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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).
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2

Quantification of miRNA and mRNA Levels

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Total RNA and miRNA from NRK-52E cells were extracted using Cell RNA Kit (19231ES50, Yeasen, China) and MolPure® Cell/Tissue miRNA Kit (19331ES50, Yeasen, China). Then, HiScript II One Step RT-PCR Kit (P612-01, Vazyme, China) was employed to react with the extract following the operating instructions. The amplification condition was set as follows: 50°C for 30 min, 94°C for 3 min, followed by 30 cycles (94°C for 30 s, 60°C for 30 s, and 72°C for 40 s), and 72°C for 5 min. The qRT-PCR was conducted in the ABI 7500 system (Applied Biosystems, USA). Relative mRNA expressions were analyzed by the 2−ΔΔCt method [25 (link)], with β-actin or U6 using for normalization. All primers sequences were shown in Table 1.
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