pcDNA3.1-FLAG- BCHE, DARS, NGDN, or UNC5B wild-type (WT) was purchased from Origene (Rockville, MD, USA), and each mutant plasmid of target genes was generated with Site-Directed Mutagenesis Kits according to the manufacturer’s protocol (Thermo Fisher Scientific). To determine the effects of each variant on cancer cell properties, GH3 cells were transfected with pcDNA3.1 control vector, pcDNA3.1-FLAG-WT of target genes, or pcDNA3.1-FLAG-mutant of target genes using Polyjet reagent (Invitrogen, Carlsbad, CA, USA). The cells were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, MTS assay (Promega, #G3582, Madison, WI, USA), and ELISA for GH (Merck, Kenilworth, NJ, USA) at 48 h after transfection42 (link).
Polyjet reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Polyjet reagent is a laboratory consumable used in the preparation of samples for analysis. It serves as a critical component in various analytical techniques, providing a standardized and consistent sample matrix. The core function of Polyjet reagent is to facilitate the homogenization and stabilization of samples, ensuring reliable and reproducible results.
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2 protocols using polyjet reagent
1
Functional Validation of Genetic Variants in Cancer Cells
2
Exogenous CTHRC1 Expression in HeLa Cells
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The plasmid pcDNA3.1-CTHRC1 was constructed by Shinegene (Shanghai, China) and the sequence of human CTHRC1 was obtained from PubMed (NM-138455.3). siRNA (1, 2 and 3) sequences were designed by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). HeLa cells (2×105) were seeded in 6-well culture plates. Plasmids (pcDNA3.1-CTHRC1 and pcDNA3.1) and siRNA were transfected into cells using PolyJet reagent according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.), when the cultures reached ~80% confluence. For pcDNA3.1 or pcDNA3.1-CTHRC1 single plasmid transfection, 1 µg of DNA, 100 µl jetPRIME buffer and 2 µl jetPRIME/well were used in a 6-well plate. Briefly, 1 µg DNA was diluted into 100 µl jetPRIME® buffer and mixed by vortexing. Then, 2 µl jetPRIME® was added, vortexed and incubated for 10 min at room temperature (RT). Transfection mix (100 µl)/well was added onto the cells, and distributed evenly by gently rocking the plates back and forth. Medium was replaced after 6 h of transfection. For plasmid and siRNA co-transfection, 1 µg DNA and 20 nM siRNA/well were used in a 6-well plate with 100 µl jetPRIME buffer and 2 µl jetPRIME. After 48 h of incubation, the cells were harvested for further analysis. Efficiency of the transfection was assessed by western blotting.
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