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Lipofectamine rnaimax

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Lipofectamine RNAiMAX is a transfection reagent designed for efficient delivery of small interfering RNA (siRNA) or microRNA (miRNA) into a wide variety of cell types. It facilitates the uptake of RNA molecules into cells, enabling effective gene silencing or modulation of gene expression.

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Lab products found in correlation

Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions) Smartpool sirna library, by Horizon Discovery (1 mentions) Smartpool sirna, by Horizon Discovery (1 mentions) X tremegene, by Roche (1 mentions) Moflo astrio, by Beckman Coulter (1 mentions) Aldefluor assay, by STEMCELL (1 mentions) Icycler iq, by Bio-Rad (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) Antisense lna gapmer, by Qiagen (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Siallstars, by Qiagen (1 mentions) Tri reagent, by Merck Group (1 mentions) Dnase 1, by Roche (1 mentions) Allstar, by Qiagen (1 mentions) On targetplus smartpool sirna, by Horizon Discovery (1 mentions) Opti mem, by Qiagen (1 mentions) Flexitube genesolution, by Qiagen (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions) One step sybr primescript rt pcr kit, by Takara Bio (1 mentions)

11 protocols using lipofectamine rnaimax

1

Genome-wide siRNA Screening for Glycolysis

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Transient siRNA reverse transfections were carried out for global siRNA screening using XTremeGene (Roche, Basel, Switzerland) according to the manufacturer's instructions with the genome-wide SmartPool siRNA library from Dharmacon using the MIA PaCa-2 HRE luciferase line. After identifying initial glycolysis genetic hits, follow-up work in each of the 3 additional cell lines listed used Lipofectamine RNAiMax (Qiagen, Valencia, CA) and Dharmacon SMARTpool siRNAs for HIF-1α, Aldolase A, AMPK, p300, PCAF, FIH, PLK-1 or the On-Target-Plus non-targeting pool #4 (OTP4). Total siRNA concentration was kept at 40 nM for single or multiple siRNA combinations. Knockdown efficiency was determined by Western blotting of cell lysates 96 hours post transfection.
Grandjean G., De Jong P., James B., Koh M.Y., Lemos R., Kingston J., Aleshin A., Bankston L.A., Miller C.P., Cho E.J., Edupuganti R., Devkota A., Stancu G., Liddington R.C., Dalby K, & Powis G. (2016). Definition of a novel feed-forward mechanism for glycolysis-HIF1α signaling in hypoxic tumors highlights adolase A as a therapeutic target. Cancer research, 76(14), 4259-4269.
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2

siRNA Transfection in 24-well Plates

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A total of 20 nM of Allstars non-silencing (Qiagen) or target gene siRNA (Qiagen) was diluted in Opti-MEM and mixed with Lipofectamine RNAiMax diluted in Opti-MEM. The RNAiMax-siRNA mixtures were incubated for 20 minutes at room temperature, followed by plating of the mixtures in 24-well plates. A total of 25,000 cells/well diluted in fully supplemented DMEM were seeded atop the RNAiMax-siRNA mixtures and incubated overnight at 37°C and 5% CO2 in a humidified incubator. Media was replaced the next day. After 72 hours of incubation, cells were collected in the appropriate buffer or fixative for downstream applications.
Jolly M.K., Ware K.E., Xu S., Gilja S., Shetler S., Yang Y., Wang X., Austin R.G., Runyambo D., Hish A.J., DeWitt S.B., George J.T., Kreulen R.T., Boss M.K., Lazarides A.L., Kerr D.L., Gerber D.G., Sivaraj D., Armstrong A.J., Dewhirst M.W., Eward W.C., Levine H, & Somarelli J.A. (2019). E-cadherin represses anchorage-independent growth in sarcomas through both signaling and mechanical mechanisms. Molecular cancer research : MCR, 17(6), 1391-1402.
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3

Identification of RELB and NFKB2 in SUM159 Cells

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Pooled small interfering RNAs for RELB (cat: L-004767-00-0005) and NFKB2 (cat: L-003918-00-0005) were obtained from GE Healthcare. SUM159 cells were seeded at a density of 500,000 cells in 10 cm cell culture dishes and allowed to adhere overnight. Transfection of 50 nM targeted siRNA and negative siRNA control (Qiagen 1027281) was performed using Lipofectamine® RNAiMAX according to manufacturer’s instructions. Eight hours following incubation plates were washed to remove excess transfection reagent. Then 72 h after initial incubation cells were harvested for flow cytometry analysis. Residual cells were stained for aldehyde dehydrogenase activity using the Aldefluor assay (Stemcell technologies) with DAPI viability dye according to manufacturer’s instruction. DEAB controls were used to gate 0.1% background for each sample tested and analyzed using a MoFlo® Astrios (Beckman Coulter).
Burnett J.P., Lim G., Li Y., Shah R.B., Lim R., Paholak H.J., McDermott S.P., Sun L., Tsume Y., Bai S., Wicha M.S., Sun D, & Zhang T. (2017). Sulforaphane enhances the anticancer activity of taxanes against triple negative breast cancer by killing cancer stem cells. Cancer letters, 394, 52-64.
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4

Investigating Estrogen Receptor Signaling

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Cells maintained in 10% Dextran-coated charcoal treated fetal bovine serum (DCC-FBS) were transfected with control or PLK1 siRNA oligonucleotides (Thermo Scientific) with Lipofectamine RNAiMAX for 48–72 h. Cells were harvested and their RNA extracted using the RNeasy Mini Kit (Qiagen). Using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), 1 μg of RNA was reverse transcribed to cDNA and real-time PCR reactions were conducted in 96-well plates using the iCycler iQ (Bio-Rad) and primers obtained from SA Biosciences (Qiagen). RNA was extracted from MCF7 and MCF7/LTED cells that had been treated with volasertib or transfected with PLK1 siRNA; cDNA was applied to the Estrogen Receptor Signaling PCR Array (SA Biosciences). Fold changes in gene expression was determined using the web-based software from SA Biosciences (http://www.qiagen.com/us/products/genes%20and%20pathways/data-analysis-center-overview-page/). Genes identified in the array were verified by qRT-PCR.
Bhola N., Jansen V.M., Bafna S., Giltnane J.M., Balko J.M., Estrada M.V., Meszoely I., Mayer I., Abramson V., Ye F., Sanders M., Dugger T.C., Van Allen E, & Arteaga C.L. (2014). Kinome wide functional screen identifies role of Polo-like kinase 1 (PLK1) in hormone-independent, ER-positive breast cancer. Cancer research, 75(2), 405-414.
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5

Knockdown and Complementation of Gene Expression

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siRNAs (see sequences in Table 1) were transfected with Lipofectamine® RNAiMAX (Life Technologies, 13778–500) for 72h at a final concentration of 30nM. All siRNAs were obtained from Dharmacon. For complementation experiments, synthetic sdRNAs (Sigma, Custom DNA and RNA Oligos) were transfected with Lipofectamine® RNAiMAX 48h after siRNA transfection. sdRNAs (Table 1) were transfected for 24h. ActB sRNAs were transfected at a final concentration of 0.5nM; Casp16p sRNAs were transfected at a final concentration of 0.25nM. Antisense LNA® GapmeRs (Qiagen) were transfected for 30h with Lipofectamine® RNAiMAX according to the manufacturer’s instructions (see sequences in Table 1). ActB and Casp16p GapmeRs were transfected at a final concentration of 50nM and 30nM, respectively. A negative control GapmeR (Qiagen) was transfected at the relevant concentration for each sRNA GapmeR. Flag-tagged hDICER WT and enzymatic-dead mutant (110 ab) plasmids (gift of Sophia Francia10 (link)) were transfected into HeLa cells at 0.75μg/mL 24h after siRNA transfection with Lipofectamine® 2000 (Life Technologies, 11668500). Transfection was performed according to the manufacturer’s instructions.
Hatchi E., Goehring L., Landini S., Skourti-Stathaki K., DeConti D.K., Abderazzaq F.O., Banerjee P., Demers T., Wang Y., Quackenbush J, & Livingston D.M. (2021). BRCA1 and RNAi factors promote small RNA- and PalB2/Rad52- mediated repair. Nature, 591(7851), 665-670.
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6

Drosha Silencing and Overexpression in HEK293 Cells

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HEK293 cells were reverse transfected with a Drosha-siRNA: 5′-GCAUGCAAGCGCGCAGUAU(dTdT)-3′ or the scrambled siRNA control siAllstars (Qiagen, Venlo, Netherlands) using Lipofectamine RNAiMAX in 6-well plates. After 24 h, cells were transfected each with 1.5 μg of plasmid encoding for YFP-Drosha-V5/His as wild-type or phosphorylation triple mutant (tripleA), pFlag/HA-DGCR8, a processing deficient TN mutant Drosha construct (TN), YFP or an EV using 6-μl PEI in 100-μl TBS buffer per well. Forty-eight hours later, RNA was isolated using TRI Reagent (Sigma-Aldrich), digested with DNaseI (Roche) and reverse transcribed. The samples were analyzed for pri-miRNA levels and endogenous Drosha mRNA levels by RT-qPCR and protein levels by western blot.
Link S., Grund S.E, & Diederichs S. (2016). Alternative splicing affects the subcellular localization of Drosha. Nucleic Acids Research, 44(11), 5330-5343.
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7

KLHL5 Knockdown via siRNA

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ON-TARGETplus SMARTpool siRNAs against Kelch-like protein 5 (KLHL5) (Horizon Discovery, Lafayette, CO) or the negative control siRNA, AllStar (Qiagen) were transfected into cells using Lipofectamine RNAiMax according to the manufacturer’s protocol. KLHL5 knockdown was verified by Western blot as described in our previous study (10 ).
Truman J.P., Ruiz C.F., Montal E., Garcia-Barros M., Mileva I., Snider A.J., Hannun Y.A., Obeid L.M, & Mao C. (2021). 1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase. Journal of Lipid Research, 63(1), 100154.
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8

Ncor1/2 Knockdown in 2iLIF Cells

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Cells were reverse transfected in 2iLIF as follows: 2.4µL of LipofectamineRNAiMAX (Life Technologies) were mixed with 200µL of OPTIMEM (Gibco) and incubated for 5 min at RT, 2.4µL of 20µM Ncor1 siRNA mix and Ncor2 siRNA mix or AllStars Neg. Control siRNA (FlexiTube GeneSolution, Qiagen) diluted in 200µL of OPTIMEM were added to the LipofectamineRNAiMAX mix and incubated for 30 min at RT, the transfection mix was added to freshly resuspended 100'000 cells in 1.6mL of medium in a well of a 12-well-plate. The next day the cells were detached and transferred to fresh 2iLIF or Serum LIF. siRNA sequences are displayed in Table S1.
Olivieri D., Papasaikas P., Lukonin I., Rittirsch M., Heß D., Smallwood S.A., Stadler M., & Betschinger J. (2020). Repression by hdac3 and dax1 mediates lineage restriction of embryonic stem cells.
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9

Chimera RNAi for EMT Transcription Factors

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Chimera RNAi for Twist-1(H00007291-R01), Snail (H00006615-R01) and Slug (H00006591-R01) and control RNAi were purchased from Novus Canada. Cells were seeded into 24-well plates at 10 5 cells per well. 30% confluent monolayer cultures were incubated with final concentration of 5 nM Chimera RNA or Negative control using Lipofectamine RNAiMAX (QIAGEN, China) for 24 h.
Ying Y., Qingwu L., Mingming X., Zhenju S., Chaoyang T, & Zhengang T. (2017). Emodin: One Main Ingredient of Shufeng Jiedu Capsule Reverses Chemoresistance of Lung Cancer Cells Through Inhibition of EMT. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 42(3).
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10

Cell Growth Profiling After ASOKD and CRISPRi

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For cell growth profiling after ASOKD, 3000 LECs per well were seeded into a 96-well plate and cultured overnight. LECs were then transfected with 20 nM of scrambled control ASO or three ASOs targeting LETR1 (Exiqon) and 0.2 µL Lipofectamine RNAiMAX previously mixed in 20 µL Opti-MEM according to the manufacturer’s instructions.
For cell growth profiling after CRISPRi, 3000 dCas9-expressing LECs per well were seeded into a 96-well plate and grown overnight. LECs were then infected with 50 MOI of lentiviruses containing vectors expressing scrambled control sgRNA or three sgRNAs targeting LETR1 diluted in complete growth EBM medium supplemented with 5 µg/mL polybrene. After 24 h, the virus-containing medium was changed.
In both experiments, LECs were continuously imaged every 3 h over three days with 4 fields per well using the IncuCyte ZOOM live-cell imaging system (Essen Bioscience). Confluence in each well was determined using IncuCyte ZOOM software (ver. 2016B). The normalized growth rate was calculated as the slope of linear regression and normalized to control. To check knockdown efficiency, LECs were harvested after 72 h post transfection. Total RNA was isolated using the RNeasy mini kit. qPCR was performed using One-Step SYBR PrimeScript RT-PCR kit (Takara Bio) on a 7900HT real-time system (Applied Biosystems).
Ducoli L., Agrawal S., Sibler E., Kouno T., Tacconi C., Hon C.C., Berger S.D., Müllhaupt D., He Y., Kim J., D’Addio M., Dieterich L.C., Carninci P., de Hoon M.J., Shin J.W, & Detmar M. (2021). LETR1 is a lymphatic endothelial-specific lncRNA governing cell proliferation and migration through KLF4 and SEMA3C. Nature Communications, 12, 925.
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