Lipofectamine rnaimax reagent

Manufactured by Thermo Fisher Scientific

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Lipofectamine RNAiMAX reagent is a lipid-based transfection reagent designed for efficient delivery of small interfering RNA (siRNA) and microRNA (miRNA) into a variety of mammalian cell types for gene silencing applications. The reagent is formulated to complex with and deliver nucleic acids into cells, enabling effective knockdown of target gene expression.

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Lipofectamine RNAiMAX (11279 citations) RNAiMAX (2357 citations) Lipofectamine (2314 citations) Lipofectamine reagent (569 citations) RNAiMAX reagent (447 citations) Lipofectamine RNAiMAX reagent protocol (8 citations) Lipofectamine RNAiMAX Transfection Reagent kit (8 citations) Lipofectamine RNAiMAX Reagent Agent (7 citations) Lipofectamine RNAiMAX siRNA transfection reagent (7 citations) Lipofectamine 2000 RNAiMAX transfection reagent (3 citations)

3 502 protocols using lipofectamine rnaimax reagent

siRNA Knockdown in HeLa Cells

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Transfection of HeLa cells was performed according to Ambion manufacturer’s guidelines (Life technologies) in 24-well culture plates using 6.25 nM siRNA and 2 μl Lipofectamine^® RNAiMAX reagent for each siRNA (Thermo Fisher Scientific) under normoxic conditions in OptiMEM I reduced serum medium (Thermo Fisher Scientific).
A mixture of two different silencer select siRNA for each cytokine (IL-6: ID: s7311, s7313; IL-8: ID: s7328, s7327; RANTES: ID: s12575, s12577) with a final concentration of 12.5 nM siRNA and 4 μl of Lipofectamine^® RNAiMAX reagent and one silencer^® select negative control (#1 siRNA) with a final concentration of 6.25 nM siRNA and 2 μl Lipofectamine^® RNAiMAX reagent and dissolved in OptiMEM to a total volume of 400 μl per well was used. At the time of transfection, cell layers were reaching a confluency of 20–30%. After an incubation time of 5 h, transfection reagents were replaced by usual HeLa growth medium without CHX for the further incubation of cells.
Knockdown efficacy was confirmed by quantitative polymerase chain reaction (qPCR) at 24, 48, 72, and 90 h post-transfection in a preliminary experiment with two silenced wells (duplicates) per time point and cytokine (see the Section “Quantitative Real-Time PCR”). Transfection time point for irradiation experiments was 24 h post-seeding and 35 h prior to infection.

Kuratli J., Pesch T., Marti H., Leonard C.A., Blenn C., Torgerson P, & Borel N. (2018). Water Filtered Infrared A and Visible Light (wIRA/VIS) Irradiation Reduces Chlamydia trachomatis Infectivity Independent of Targeted Cytokine Inhibition. Frontiers in Microbiology, 9, 2757.

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Visualizing Nuclear Actin and Emerin Dynamics

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Cells were transfected using Lipofectamine 2000 reagent (Life Technologies, Cat. 11668030). Nuclear actin was visualized by transfecting cells with Actin-Chromobody^® plasmid (Actin-nanobody, fused to TagGFP containing a nuclear localization sequence, (ChromoTek). Emerin-pEGFP-C1 plasmid was obtained from Addgene, plasmid #61993^{55 (link)}. Dominant-negative KASH fused with EGFP plasmid was previously reported^{56 (link),57 (link)}.
RNAi to target EMD, MAN1 and LAP2α was performed using Lipofectamine RNAiMAX reagent (Life Technologies, Cat. 13778030) and 10 nM Human gene specific 27mer siRNA duplexes (Origene) with following sequences:
EMD: ‘rCrCrArArGrArArArGrArGrGrArCrGrCrUrUrUrArCrUrCTA’
MAN1 (LEMD3): ‘rCrUrGrUrUrGrArUrArUrArUrArArUrUrGrUrCrArGrUrCCA’
LAP2α (TMPO): ‘rGrArUrArArArCrCrCrArGrArCrArArGrArArGrArUrArAAG’
RNAi to target LMNB1 was performed using Lipofectamine RNAiMAX reagent (Life Technologies, Cat. 13778030) and 10 nM human gene specific siRNA duplexes (abx903005, Abbexa Ltd) with following sequence:
LMNB1:‘GCAGACUUACCAUGCCAAATTUUUGGCAUGGUAAGUCUGCTT’
As a control, a Trilencer-27 Universal Scrambled Negative Control siRNA Duplex (Origene) was used. Cells were visualized after 48 h post-transfection.

Nastały P., Purushothaman D., Marchesi S., Poli A., Lendenmann T., Kidiyoor G.R., Beznoussenko G.V., Lavore S., Romano O.M., Poulikakos D., Lagomarsino M.C., Mironov A.A., Ferrari A, & Maiuri P. (2020). Role of the nuclear membrane protein Emerin in front-rear polarity of the nucleus. Nature Communications, 11, 2122.

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MiR-21 Mimics and Inhibitors Transfection

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The synthetic miR-21 mimics and miR-21 inhibitors were purchased from RiboBio (Guangzhou, People’s Republic of China). All mimics and inhibitors contain 2′-O-methyl modification at each base. Stealth™ RNAi (Life Technologies) was used as a negative control. Lipofectamine™ RNAiMAX reagent (Life Technologies) was used for the transfection of DBTRG cells. Briefly, DBTRG cells (0.5−1×10⁶ cells/well in 6-well plates with 2 mL volume) were cultured in DMEM supplemented with 10% FBS for 24 h. MiR-21 mimics and inhibitors or negative controls (25 pmol) in 150 μL Opti-MEM medium (Life Technologies) were mixed with the same volume of Opti-MEM medium containing 9 μL of Lipofectamine RNAiMAX reagent and incubated at room temperature for 5 min to allow the formation of transfection complexes. The transfection complexes were added to the appropriate cell cultures by gently swirling the plates. The culture medium was replaced with fresh medium after 5–6 h and the cells were continuously incubated for 48 h.

Tang X.J., Huang K.M., Gui H., Wang J.J., Lu J.T., Dai L.J., Zhang L, & Wang G. (2016). Pluronic-based micelle encapsulation potentiates myricetin-induced cytotoxicity in human glioblastoma cells. International Journal of Nanomedicine, 11, 4991-5002.

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siRNA and miRNA Transfection Protocol

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Small interfering RNA (siRNA) duplexes targeting PAK4, PAK4 siRNA 1 (D-003615-06-0020) and 2 (D-003615-07-0020), and non-targeting (NT) siRNA were purchased from Dharmacon (Lafayette, CO). Precursors of respective human microRNAs (miR-27a, -122, -128, -193 and -217) and negative controls were purchased from Life Technologies, Thermo Fisher Scientific, CA.
Transfections were performed with Lipofectamine RNAiMAX Reagent (Life Technologies). For si/miRNA transfections, VM-CUB1 and RT-112 cells were seeded at 1 × 10 5 cells per well in 6well plates along with siRNA duplexes or miRNAs using Lipofectamine RNAiMAX Reagent (Life Technologies). At 72 hours after transfection, cells were either seeded for cell proliferation, colony formation, and invasion assays or were harvested for RNA isolation or immunoblot analysis.

Chandrashekar D.S., Chakravarthi B.V.S.K., Robinson A.D., Anderson J.C., Agarwal S., Balasubramanya S.A.H., Eich M.L., Bajpai A.K., Davuluri S., Guru M.S., Guru A.S., Naik G., Della Manna D.L., Acharya K.K., Carskadon S., Manne U., Crossman D.K., Ferguson J.E., Grizzle W.E., Palanisamy N., Willey C.D., Crowley M.R., Netto G.J., Yang E.S., Varambally S, & Sonpavde G. (2020). Therapeutically actionable PAK4 is amplified, overexpressed, and involved in bladder cancer progression. Oncogene, 39(20).

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Gene Knockdown Assay with Scrambled and Targeted siRNAs

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For the gene knock-down assay, scrambled siRNA as negative control (SIC-001-10, MISSION siRNA Universal Negative Control) [26 (link)] or siRNA for ATP8A2 (SASI_Mm02_00323184) were purchased from Sigma. C6 glioma cells were transfected by Lipofectamine™ RNAiMAX reagent (Life Technologies Corp., Tokyo, Japan). Transfected cells were cultured for 72 h at 37 °C prior to use in experiments. siRNAs for syntaxin-1A (Stx-1A) (SASI_Rn01_00065142) and annexin A2 (ANXA2) (SASI_Rn01_00033819) were purchased from Sigma. Primary cultured cortical neurons (4 days in vitro) were transfected by Lipofectamine™ RNAiMAX reagent (Life Technologies Corp., Tokyo, Japan). Transfected cells were cultured for 72 h at 37 °C prior to use in experiments.

Matsunaga H., Halder S.K, & Ueda H. (2021). Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions. Cells, 10(3), 567.

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Bovine Embryo Transfection via WOW Culture

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Bovine one-cell embryos were produced in vitro as previously described elsewhere [9 (link)]. At 20 h post-insemination (hpi), the embryos
were treated with pronase solution (0.5% [w/v] in PBS[+] containing 5.56 mM glucose and 0.5 mM sodium pyruvate) to remove the zona pellucida. The zona-free one-cell embryos were individually
allocated to a WOW system [7 (link), 8 (link)] using the LinKID micro25 culture dish (Dai Nippon Printing, Tokyo, Japan) with 50
µl of modified synthetic oviduct fluid (mSOF) [12 (link)] and cultured until 72 hpi (day 3). At 72 hpi, only the embryos that had developed to the 8-16-cell
stage were retained. siRNA lipofectant was prepared using the mSOF and Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions with
some modifications, resulting in a lipofectant that was composed of mSOF containing 50–125 nM siRNA and 0.3% (v/v) of Lipofectamine RNAiMAX reagent. The 8-16-cell stage embryos were
transfected with siRNA by replacing the culture medium with 50 µl of the lipofectant and further cultured up to 192 hpi. All the cultures were performed at 38.5°C under 5% CO₂, 5%
O₂ and 90% N₂.

IKEDA S., SUGIMOTO M, & KUME S. (2018). Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system. The Journal of Reproduction and Development, 64(2), 199-202.

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siRNA Knockdown of ARMC5, SREBF2, and CUL3

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Silencer Select siRNAs (12 pmol) targeting to ARMC5 (Thermo Fisher Scientific, s36352), SREBF2 (Thermo Fisher Scientific, s29) or Silencer Select Negative Control No.1 siRNA (Thermo Fisher Scientific) were introduced to NCI-H295R cells by reverse transfection using 3 μL of Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) per 12-well plate according to the protocol provided by the manufacturer. Silencer Select siRNA (180 pmol) targeting to CUL3 (Thermo Fisher Scientific, s16050) or Silencer Select Negative Control No.1 siRNA (Thermo Fisher Scientific) were introduced to HEK293T cells using 30 μL of Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) per 10 cm dish according to the protocol provided by the manufacturer.

Okuno Y., Fukuhara A., Otsuki M, & Shimomura I. (2022). ARMC5-CUL3 E3 ligase targets full-length SREBF in adrenocortical tumors. JCI Insight, 7(16), e151390.

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miRNA Transfection in Endothelial Cells

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The ECs grown to 70%–90% confluence were transfected with 5 nM miR-200b stem-loop precursor (PM10492, stem-loop accession number: miRBase: MI0000342), or 5 nM miR-466 stem-loop precursor (PM18443, stem-loop accession number: miRBase: MI0014157), or 5 nM Pre-miR miRNA Precursor Negative Control (AM17110, all purchased from Thermo Fisher Scientific) by using Lipofectamine RNAiMAX Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. For the inhibition of miRs, ECs were transfected with miR-200b inhibitor (AM10492, stem-loop accession number: miRBase: MIMAT0000318), or miR466 inhibitor (AM18443, stem-loop accession number: miRBase: MI0014157), or Anti-miR miRNA Inhibitor Negative Control #1 (AM17010, all from Thermo Fisher Scientific) by using Lipofectamine RNAiMAX Reagent. Cells were incubated at 37°C for 48–72 h before being harvested for experiments.

Kujawa M., O’Meara M., Li H., Xu L., Meda Venkata S.P., Nguyen H., Minjares M., Zhang K, & Wang J.M. (2022). MicroRNA-466 and microRNA-200 increase endothelial permeability in hyperglycemia by targeting Claudin-5. Molecular Therapy. Nucleic Acids, 29, 259-271.

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Optimizing siRNA Transfection for Cell Viability

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The siRNA transfection was performed with reverse transfection method using Lipofectamine RNAi MAX reagent (Thermo Fisher Scientific). Briefly, siRNA at different concentrations was diluted in 10 μl Opti-MEM (Thermo Fisher Scientific, USA) and mixed with 10 μl Opti-MEM containing 0.05 μl Lipofectamine RNAi MAX reagent, and the transfection mixture was incubated at room temperature for 5 min. Cell solutions (3,000 cells per well in a 96-well plate) were added to each well containing the transfection mixture. Cells were then incubated for 72 h, and cell images were taken with IncuCyte ZOOM (Essen BioScience, Ann Arbor, MI). Cell viability was assessed with AlamarBlue assay, and the siRNA sequences are shown in Supplementary Table 1.

Ren G., Chen J., Pu Y., Yang E.J., Tao S., Mou P.K., Chen L.J., Zhu W., Chan K.L., Luo G., Deng C, & Shim J.S. (2024). BET inhibition induces synthetic lethality in PTEN deficient colorectal cancers via dual action on p21CIP1/WAF1. International Journal of Biological Sciences, 20(6), 1978-1991.

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RNA Interference-Mediated Knockdown Optimization

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Cells (150–200 × 10³) were plated in 35 mm plates and transfected 12 hrs later with the siRNA against the target selected—except for METTL3, where a mix of four siRNAs was used—or the negative control (final concentration 30 nM) using 5 μl of Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) and 300 μl of Opti-MEM (Thermo Fisher Scientific). The medium was replaced 5–12 hrs later. Cells were harvested 48 hrs later or used for further analyses. For FACS analysis, cells (600 × 10³) were plated in 60 mm plates and transfected 12 hrs later with the siRNA against the target selected or the negative control (final concentration 30 nM) using 10 μl of Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) and 600 μl of Opti-MEM (Thermo Fisher Scientific). Cells were passed 5 hrs later to a 100 mm plate and collected 48 hrs later. For the double knock-down of YTHDC1 and DDX5, a combination of two different siRNA was used at a final concentration of 60 nM. Cells were passed 5 hrs later to a 100 mm plate and collected 72 hrs later. For actinomycin D treatment, cells depleted for YTHDC1 and DDX5 were split into two different plates and, after 12 hrs, harvested or kept in their medium added with actinomycin D (5 mg/ml, Sigma-Aldrich) for 6 hrs or 12 hrs.

Dattilo D., Di Timoteo G., Setti A., Giuliani A., Peruzzi G., Beltran Nebot M., Centrón-Broco A., Mariani D., Mozzetta C, & Bozzoni I. (2023). The m6A reader YTHDC1 and the RNA helicase DDX5 control the production of rhabdomyosarcoma-enriched circRNAs. Nature Communications, 14, 1898.

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