Respective siRNAs were synthesized by GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China). The following siRNA sequences were used: Sense: 5′-GCUCCUUCUCAAUCCGCCUTT-3′, Antisense: 5′-AGGCGGAUUGAGAAGGAGCTT-3′; siNC: Sense 5′-UUCUCCGAACGUGUCACGUTT-3′, Antisense 5′-ACGUGACACGUUCGGAGAATT-3′. A total of 3×105 OS cells were seeded per well in a 6-well plate, and transiently transfected with 10 nM siRNA using Lipofectamine iMAX kit (Invitrogen), according to the manufacturer's instructions. OS cells were harvested for RNA and protein extraction after 48-72 hours of incubation with respective siRNAs.
Lipofectamine imax kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Lipofectamine iMAX kit is a lipid-based transfection reagent designed for efficient delivery of nucleic acids, such as plasmid DNA, mRNA, and siRNA, into a variety of eukaryotic cell types. The kit includes the Lipofectamine iMAX transfection reagent and the necessary buffers and solutions for the transfection process.
Automatically generated - may contain errors
5 protocols using lipofectamine imax kit
1
siRNA-mediated Knockdown of Target Genes
2
Silencing MXI1 in Osteosarcoma Cells
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Human osteoblast hFOB1.19 and two human osteosarcoma cells Saos-2 and U2OS (ATCC, United States) were maintained in DMEM plus 10% heat-inactivated fetal bovine serum (Sigma, United States) and 2% antibiotic-antimycotic solution (Corning, United States) at 37°C in a humidified atmosphere with 5% CO2. Specific siRNAs against MXI1 (si-MXI1) and negative control (si-NC) were synthesized by GenePharma (Shanghai, China). In total, 3 × 105 Saos-2 and U2OS cells were seeded onto a 6-well plate, and transiently transfected with 10 nM siRNA utilizing Lipofectamine iMAX kit (Invitrogen, United States). Saos-2 and U2OS cells were collected for RNA extraction following 48 h of incubation with specific siRNAs.
3
Generating Functional circRNA Constructs
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Human circFUT8 linear sequence was obtained from LUAD tissues by PCR and inserted into plasmid vector pcDNA 3.1 (Hanbioa). The small interfering RNA (siRNA) of circFUT8, mFUT8 were provided by RiboBio. The target sequences are supplied in Table S1 . According to the target sequence of si‐circFUT8, cloned a short hairpin RNA (shRNA) (sh‐circFUT8) into pGFP‐u6 vector. A Lipofectamine 3000 kit (Invitrogen) was used to perform transient transfection of the shRNA or overexpressing plasmids, and the Lipofectamine iMax kit (Invitrogen) was used to perform transient transfection of siRNA.
4
Transfection of MIR22HG and STAT3 siRNA
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Transfections were performed using the Lipofectamine iMAX kit (Invitrogen) according to the manufacturer’s instructions. The siRNAs of MIR22HG or STAT3 and scrambled siRNA (siCtrl) were purchased from Dharmocom. After 48-72 hours incubation with siRNAs (10 nM), cells were harvested for RNA and protein extraction.
5
Modulating circDCUN1D4 and Associated Genes
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shRNAs targeting the junction region of the circDCUN1D4 sequence, circDCUN1D4-overexpressing plasmids, HuR-overexpressing plasmids, and TXNIP-overexpressing plasmids were synthesized by Hanbio (Shanghai, China). The primers are provided in Table S2 . The siRNAs targeting TXNIP, DHX9, and ADAR1 were provided by RiboBio (Guangzhou, China). The target sequences are supplied in Table S3 . The sequences of AluJo + AluSc, AluJo, AluSc, and vector were constructed by sequencing synthesis and subcloned into pcDNA3.1(+) (Public Protein/Plasmid Library, Nanjing, China). Transient transfection of the shRNA or the overexpressing plasmids was performed using the Lipofectamine 3000 kit (Invitrogen), according to the manufacturer’s instructions, and transient transfection of siRNA was performed using the Lipofectamine iMax kit (Invitrogen), according to the manufacturer’s instructions.
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