Agarose gel extraction kit

The total RNA of MMGZ01 hybridoma cell line was isolated using a Trizol reagent (Invitrogen, Burlington, ON, Canada), and reverse-transcribed to cDNA with a M-MuLV RT-PCR kit (Sangon Biotech, Shanghai, China). In the Polymerase Chain Reaction (PCR), PrimeSTAR HS DNA polymerase was used to amplify the VH and VL genes with designed degenerate primers specific for murine antibodies (Takara Bio, Dalian, Liaoning, China). Chains of VH and VL were detected in a 1% agarose gel and corresponding bands were purified using an Agarose Gel Extraction Kit (Takara Bio, Dalian, Liaoning, China). The purified chains were then cloned to pMD18T vector for sequencing (Genscript Corporation, Nanjing, Jiangsu, China).

Messenger RNA was purified from total RNA, which was isolated from 5 × 10⁶ hybridoma cells, following the instructions of the manufacturer of the UNIQ-10 Column Trizol Total RNA Isolation Kit and the mRNA purification kit (Sangon Biotech, Shanghai, China). First-strand cDNA was synthesized from mRNA using SuperScriptIII reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The antibody variable genes were amplified from cDNA via PCR using appropriate degenerate oligonucleotide primer sets (see Table 3) with the FastPfu DNA polymerase. The PCR product was analyzed via 1.0% (w/v) agarose gel electrophoresis and visualized by adding GelRed stains (Biotium, Hayward, CA, USA). A unique band at approximately 400 bp indicated a positive result.
After purification using an agarose gel extraction kit (Takara Biotechnology, Shiga, Japan), the VH and VLλ genes were separately cloned into the pEASY-Blunt Zero vector and then transformed into the E. coli strain Trans1-T1 at 42 °C for 30 s. The transformants were selected on Luria Bertani plates supplemented with 50 μg·mL⁻¹ ampicillin and kanamycin. After overnight incubation at 37 °C, the plasmids from the culture of colonies were extracted using a mini preparation kit (Axygen, Beijing, China). The positive plasmids were confirmed via PCR and DNA sequencing using the M13 universal primers.

ABI7500 real-time polymerase chain reaction (PCR) instrument (Applied Biosystems Inc., Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). A NanoPhotometer nucleic acid and protein ultraviolet detector (NanoPhotometer^® Pearl; Implen GmbH, Munich, Germany) and a 3K18 type low temperature high speed centrifuge (Sigma, Osterode am Harz, Germany) were used. The UVP GelDoc-It 310 gel imaging analysis system was purchased from Shanghai Kunke Co., Ltd. (Shanghai, China). TRIzol reagent, LA Taq DNA polymerase and lipid Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) were used. The miRNeasy Mini kit serum total RNA extraction kit was from QIAGEN Inc. (Hilden, Germany). For cell culture, 10% FBS RPMI 1640 medium and DMEM culture medium (Hyclone, GE Healthcare, Little Chalfont, UK) were used. Agarose gel extraction kit and mir-21qPCR primer kit were purchased from Takara Bio Inc. (Otsu, Japan). Lentiviral vector LV-anti-miR-21 and control vector were from Shanghai SBO Medical Biotechnology Co. (Shanghai, China). SPRY2 eukaryotic expression vector was purchased from Origene (Rockville, MD, USA) and microRNA-21 mimics and inhibitors were from Biomics Biotechnologies (Nantong) Co., Ltd. (Nantong, China).