Lipofectamine rnaimax

Manufactured by GenePharma

Sourced in China

Lipofectamine RNAiMAX is a transfection reagent designed for efficient and reliable delivery of small interfering RNA (siRNA) and other nucleic acids into a wide range of cell lines. It is formulated to facilitate the uptake of RNA molecules into the target cells, enabling the study of gene function through RNA interference (RNAi) techniques.

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18 protocols using lipofectamine rnaimax

Transfection of HEK293T cells with siRNA and plasmids

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A mixture of Lipofectamine RNAimax and siRNA (GenePharma, Suzhou, Jiangsu, China) was added to HEK293T cells in Opti-MEM. The medium was changed to DMEM after 4–6 h of culture, and the cells were incubated at 37 °C in a 5% CO₂ incubator. Forty-eight hours later, the siRNA was expressed successfully and could be used for further experiments. The siRNAs used are listed in Supplementary Table S1.
The constructed plasmids (GeneRay, Beijing, China) were premixed with Lipofectamine 2000 in Opti-MEM (Thermo) and then applied to the cells. The medium was changed to DMEM after 4–6 h. The cells were incubated at 37 °C in a 5% CO₂ incubator for 48 h prior to further experiments.

Zeng K.W., Wang J.K., Wang L.C., Guo Q., Liu T.T., Wang F.J., Feng N., Zhang X.W., Liao L.X., Zhao M.M., Liu D., Jiang Y, & Tu P. (2021). Small molecule induces mitochondrial fusion for neuroprotection via targeting CK2 without affecting its conventional kinase activity. Signal Transduction and Targeted Therapy, 6, 71.

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Knockdown of SMC4 Expression

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For the knockdown experiments, the cells were seeded in six-well plates and transfected with siRNA (small interfering RNA) using the Lipofectamine RNAiMAX transfection reagent (GenePharma, Shanghai, China) according to the manufacturer’s instructions. A study has shown that SMC4-Homo-830 greatly decreases the expression of SMC4 (18 (link)). SMC4-Homo-830 was cloned into the pGPU/GFP/Neo-shNC (RNAi control vector containing GFP gene, and includes neomycin resistant gene) vector (GenePharma) and transfected into the cell lines.

Zhang S.R., Li J., Chen J.X., Chen G., Chen J.Y., Fu H.W, & Zhou B. (2022). SMC4 enhances the chemoresistance of hepatoma cells by promoting autophagy. Annals of Translational Medicine, 10(24), 1308.

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Transfection of HCT116 Cells with siRNA Oligos

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Transfection of HCT116 cells was conducted with Lipofectamine RNAiMAX (GenePharma, Shanghai, China) following the manufacturer's instructions. High purity controls (scrambled RNA), along with DR5, p53, CHOP, Atg7 and JNK siRNA oligos, were obtained from GenePharma. The targeting sequences of the siRNA constructs are: DR5 siRNA-1, 5′-UUCUGGGAACACGAGCAACAG-3′ DR5 siRNA-2, 5′-UUUAGCCACCUUUAUCUCAUUGUCC-3′ p53 siRNA-1, 5′-AAGACUCCAGUGGUAAUCUAC-3′ p53 siRNA-2, 5′-CGGCAUGAACCGGAGGCCCAU-3′ CHOP siRNA-1, 5′-UUCUUGGUCGUCUCCAGUGUU-3′ CHOP siRNA-2, 5′-GCCUGGUAUGAGGACCUGC-3′ Atg7 siRNA-1, 5′-GGAGUCACAGCUCUUCCUU-3′ Atg7 siRNA-2, 5′-GGAACACUGUAUAACACCA-3′ JNK siRNA-1 is a combination of two sequences including 5′-UUCGGGUCAUUAUAUCAUCAU-3′ and 5′-GGCACUUGAGCUGGUGAAUUU-3′ JNK siRNA-2, 5′-UUUUUCUUACAGGAUGGAAGA-3′.

Chen L., Meng Y., Sun Q., Zhang Z., Guo X., Sheng X., Tai G., Cheng H, & Zhou Y. (2016). Ginsenoside compound K sensitizes human colon cancer cells to TRAIL-induced apoptosis via autophagy-dependent and -independent DR5 upregulation. Cell Death & Disease, 7(8), e2334-.

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Transfection and Plasma Treatment of Stem Cells

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SCs were cultured in Opti-MEM^® reduced serum medium without antibiotics one day before transfection. At 60–80% confluence, SCs were mock-transfected (Lipofectamine^® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine^® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol. The sequence of the miR-7450 agomir was 5′- UCUGUUCUUAAGGAGGCUGAGGC-3′ and 5′-CUCAGCCUCCUUAAGAACAGAUU-3′. The sequence of the miR-7450 antagomir was 5′-GCCUCAGCCUCCUUAAGAACAGA-3′. The sequence of the miR-7450 agomir NC was 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′. The sequence of the miR-7450 antagomir NC was 5′-CAGUACUUUUGUGUAGUACAA-3′. Transfected SCs were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 6 h. The medium was replaced with fresh Opti-MEM^® medium, and the cells were incubated for a further 48 h.

Zhang J.J., Wang X.Z., Luong Do H., Chandimali N., Kang T.Y., Kim N., Ghosh M., Lee S.B., Mok Y.S., Kim S.B., Kwon T, & Jeong D.K. (2018). MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation. Scientific Reports, 8, 8761.

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miR-320a Modulation in ER Stress

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Cells were seeded in 6-well and 12-well plates at a density of 1.25x10⁵-5x10⁵ cells per well and transfected with miR-320a mimic (Qiagen, 339173 YM00471432) or antagomir (GenePharma) 18–24 hours after seeding with Lipofectamine RNAiMax. A plasmid containing endogenous miR-320a gene was transfected with polyethylenimine (PEI) (Polysciences Inc, 24765–1) [19 (link)]. The medium was replenished 12 hours after transfection. 50 nM thapsigargin, 2 μg/mL tunicamycin, or 2 mM histidinol was added 24 hours after transfection and incubated for the designated time before being collected for western blot.

Fields C.J., Li L., Hiers N.M., Li T., Sheng P., Huda T., Shan J., Gay L., Gu T., Bian J., Kilberg M.S., Renne R, & Xie M. (2021). Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a. PLoS Genetics, 17(12), e1009934.

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Kdm7a Modulation in Cell Differentiation

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The expression construct of Kdm7a was obtained from Origene (Rockville, MD, USA). A mutant mouse Kdm7a construct with H282A mutation that abolishes the enzymatic activity of KDM7A26 was made by using a mutagenesis kit (Vazyme Biotech, Nanjing, China). For the Kdm7a loss of function studies, we transfected ST2 cells with either 30 nmol/L Kdm7a siRNA or negative control siRNA (Genepharma, Shanghai, China) using lipofectamine RNAi‐Max (Gaithersburg, MD, USA). For the Kdm7a gain‐of‐function experiments, the ST2 cells were transfected with wild‐type or mutant Kdm7a expression plasmid, or the empty vector using Attractene transfection reagent (QIAGEN, Hilden, Germany) for 16 hours. Adipogenic or osteogenic induction was performed at appropriate confluence to allow the cells to differentiate.
For the co‐transfection studies, the Kdm7a expression plasmid and Sfrp1 siRNA were co‐transfected by using Attractene transfection reagent for 16 hours. At appropriate confluence of cells, adipogenic or osteogenic induction was performed to allow the cells to differentiate.

Yang X., Wang G., Wang Y., Zhou J., Yuan H., Li X., Liu Y, & Wang B. (2019). Histone demethylase KDM7A reciprocally regulates adipogenic and osteogenic differentiation via regulation of C/EBPα and canonical Wnt signalling. Journal of Cellular and Molecular Medicine, 23(3), 2149-2162.

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LEMD1 Knockdown and Overexpression in Thyroid Cancer

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The negative control small interfering RNA (siRNA), the LEMD1-specific siRNA used for LEMD1-knockdown, the empty control vector [pEX-3 (PGCMV/MCS/Neo)] and the LEMD1 overexpression vector [pEX-3 (EcoRI/BamHI)-LEMD1] were purchased from Shanghai GenePharma Co., Ltd. The siRNA (20 µM/µl) was transfected into TC cell lines using Lipofectamine RNAiMAX (cat. no. 13778075; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol for 8 h at 37°C. The vectors (1 µg/µl) were transfected into TC cell lines using Lipofectamine 3,000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) for 6 h at 37°C. The TC cells were seeded into 6-well plates and incubated at 37°C for 24 h before transfection (TPC-1, 4.0×10⁴/well; BCPAP and KTC-1, 8.0×10⁴/well). Subsequently, the transfected TC cells were incubated at 37°C for 48 h before being harvested for subsequent experimentation. The siRNA sequences were as follows: siRNA-LEMD1 sense, 5′-GCCCAAUACUACCUUCCACTT-3′ and antisense, 5′-GUGGAAGGUAGUAUUGGGCTT-3′; and the non-targeting negative control siRNA sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. The transfection efficiency was tested using RT-qPCR, as aforementioned. All experiments were performed in triplicate with three independent samples.

Xu M., Lin B., Zheng D., Wen J., Hu W., Li C., Zhang X., Zhang X, & Qu J. (2021). LEM domain containing 1 promotes thyroid cancer cell proliferation and migration by activating the Wnt/β-catenin signaling pathway and epithelial-mesenchymal transition. Oncology Letters, 21(6), 442.

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CDKN2A Knockdown Impacts BM-MSC Proliferation

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Since the CDKN2A might be important in proliferation activity in patient's BM‐MSCs, we performed CDKN2A KD experiment and analysed proliferation activity using a real‐time cell monitoring system. CDKN2A KD was performed by small interfering RNA (siRNA) with specific sequence of 5′‐CCGUAAAUGUCCAUUUAUATTUAUAAAUGGACAUUUAUGGTT‐3′ designed and synthesized by GenePharma (Shanghai GenePharma, Shanghai, China). BM‐MSCs at third passage (1.0 × 10⁴) were suspended in 150 μl of antibiotic‐free basic cell culture medium and seeded into each well of the E‐plate 16 (ACEA Biosciences Inc, San Diego, USA) and then installed into xCELLigence RTCA DP system (ACEA Biosciences Inc). After 24 hr, cells were transfected with 50 nM CDKN2A‐siRNA using Lipofectamine RNAiMAX (Invitrogen Life Technologies, Carlsbad, CA, USA). Untreated, Lipofectamine RNAiMAX treated and negative control siRNA (no genetic homology with human siRNA, GenePharma) treated cells were used as controls. The proliferation activity was measured every 10 min for following 3 days and analyses using RTCA software 2.0 (ACEA Biosciences Inc).

Choi H., Kim Y., Kang D., Kwon A., Kim J., Min Kim J., Park S., Kim Y., Min C, & Kim M. (2020). Common and different alterations of bone marrow mesenchymal stromal cells in myelodysplastic syndrome and multiple myeloma. Cell Proliferation, 53(5), e12819.

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Transfection and Oxygen-Glucose Deprivation in Cell Lines

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HL-60 cells were cultured in 1640 RPMI medium containing 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin and incubated in a humidified incubator at 37°C with 5% CO₂ at 37°C. HL-60 cells were transfected with a mixture of miR-29b agomir/control and Lipofectamine RNAiMAX (GenePharma) before a further 24-hour incubation in a humidified incubator. Human brain capillary endothelial cells (hCMEC/D3) were cultured in EBM-2 medium supplemented with vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), basic FGF, hydrocortisone, ascorbate, penicillin-streptomycin, and 2.5% FCS. hCMEC/D3 in the OGD group was kept in an ischemia-mimetic solution and kept for 2.5 hours at 37°C in a hypoxic incubator chamber filled with 94.5% N₂, 0.5% O₂, and 5% CO₂. hCMEC/D3 was then transferred to normal culture medium for 24 hours and kept at 37°C in an incubator with 5% CO₂ for reoxygenation. The hCMEC/D3 cells were divided into three groups: sham+control-HL-60 group, OGD+control-HL-60 group, and OGD+miR-29b agomir-HL-60 group (20 nM).

Ma X., Yun H.J., Elkin K., Guo Y., Ding Y, & Li G. (2022). MicroRNA-29b Suppresses Inflammation and Protects Blood-Brain Barrier Integrity in Ischemic Stroke. Mediators of Inflammation, 2022, 1755416.

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Transfection of Cardiac Microvascular Endothelial Cells

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The isolated CMECs were cultured to 80% confluence with complete culture medium as described above at 37 °C in a 5% CO₂ and 95% air incubator and then transfected with rno-miR-21-5p mimics (5'-UAGCUUAUCAGACUGAUGUUGA-3'; 100 nM; GenePharma) or the scrambled control (5'-UUCUCCGAACGUGUCACG-3'; 100 nM; GenePharma) using Lipofectamine RNAiMAX (cat. no. 13778150; Invitrogen). The transfected cells were used for qPCR, cell viability assays, migration assays, tube formation assays, apoptosis assays or western blot assays. In the above assays, 3 identical wells were observed in each analysis, and three repeat experiments were conducted.

Liao Z., Chen Y., Duan C., Zhu K., Huang R., Zhao H., Hintze M., Pu Q., Yuan Z., Lv L., Chen H., Lai B., Feng S., Qi X, & Cai D. (2021). Cardiac telocytes inhibit cardiac microvascular endothelial cell apoptosis through exosomal miRNA-21-5p-targeted cdip1 silencing to improve angiogenesis following myocardial infarction. Theranostics, 11(1), 268-291.

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