Lipofectamine reagents go online to view related products. Lipofectamine rnaimax, what medium for transfection. The hdr template the hdr template contains a 6base eco ri recognition site that enables assessment of hdr efficiency by. Lipofectamine 3000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to transfect cells. Can anybody give me exact protocol for sirna transfection. Engen spy cas9 nls, is an rnaguided endonuclease that catalyzes site specific cleavage of double stranded dna. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Transfection of cyp3a4targeting sirna using either promofectinhepatocyte or lipofectamine rnaimax in hepatopac cocultures resulted in functional knockdown of cyp3a4.
The transfection reagents lipofectamine rnaimax and lipofectamine 3000 showed the best performance in. Lipofectamine 2000 and lipofectamine rnaimax transfection. I am using the standard protocol for lipofectamine rnaimax plate cells to 6080% confluency, dilute 9l lipofectamine in 150l optimem, dilute 30 pmol sirna in 150l optimem. Download lipofectamine rnaimax reagent protocol pdf. This study has shown for the first time that lipofectamine rnaimax is a suitable transfection reagent, thereby justifying it as the correct preference for selectively silencing topo ii. Due to the multitude of reagents and cell types, rnai experiment. Livecell imaging to compare the transfection and gene. Download altogen biosystems c2c12 transfection protocol.
Superior knockdown with lipofectamine rnaimax transfection reagent compared to competing sirna transfection reagents. The pam sequence, ngg, must follow the targeted region on the opposite strand of the dna with respect to the region complementary sgrna sequence. The lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna, and lipofectamine messengermax reagent delivers mrna. This is a reverse transfection method that uses a final concentration of 10 nm rnp per transfection in a 96well culture plate. High throughput rnai assay optimization using adherent cell. Citations are the number of other articles citing this article, calculated by crossref and updated daily.
Mar 08, 2008 rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. Original article an improved method for increasing the. Disregarded effect of biological fluids in sirna delivery core. Download lipofectamine rnaimax reagent protocol doc.
Cells were transfected with lipofectamine rnaimax complexed with silencer select sirna at 30 nmwell. Rnaimax reverse transfections lipofectamine thermo fisher. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types. Transient transfection of mammalian cells with lipofectamine. Mix gently and incubate for 1020 minutes at room temperature. Both lipofectamine rnaimax and dhmafect fail to give satisfied gene silencing on hepg2 cells from final 5. Here we present a method for the transfection of cas9 rnps into hek293 ft cells using thermo fisher lipofectamine rnaimax.
For each well to be transfected, prepare rnai duplex lipofectamine rnaimax complexes as follows. Tube a contains optimem media and rnaimax reagent and tube b contains optimem media and sirna. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirna mediated gene. Due to the multitude of reagents and cell types, rnai experiment optimization can be timeconsuming. Lipofectamine rnaimax for your cotransfections, perform a reverse transfection as described on page 2 with the following modifications. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Jul 26, 2016 there are several ways in which to introduce cas9guide rna complexes into cells. Lipofectamine rnaimax transfection reagent thermofisher.
L final component per well 384well 96well 24well sirna 30 nm final 1. High throughput rnai assay optimization using adherent. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. See the engen sgrna synthesis kit manual for further details. The cell density per well and amount of lipofectamine rnaimax used per well were as follows. Lipofectamine rnaimax reagent thermo fisher scientific ca. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. In this study, we used three transfection reagents lipofectamine rnaimax, oligofectamine and lipofectamine 2000 to deliver sirna into human embryonic stem hes. Automated highthroughput sirna transfection in raw 264. Amounts of reagent and sirna can be scaled up or down depending on the number of cells that will be transfected. K65p mimic mirvana with lipofectamine rnaimax thermofisher scientific. Comparison of small interfering rna sirna delivery into. Add the rnai duplex lipofectamine rnaimax complexes to each well.
Lower confluence will achieve higher percentage of transfection but may yield a lower absolute number of transfected cells. In this study adherent cell cytometry was used to rapidly optimize sirna transfection in human aortic vascular smooth muscle cells aosmc. Methods aosmc were seeded at a density of 30008000 cellswell of a. The efficiencies of genome modification were similar among three target loci in dna, mrna and cas9 proteintransfected cells fig. Rapid and highly efficient mammalian cell engineering via. Lipofectamine rnaimax, a new transfection reagent, has been confirmed high efficiency in delivering small interfering rna sirna into mesenchymal stem cells and neural stem cells.
Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Human ascites fluid severely restricts cellular uptake. A case study for optimization procedure jeanphilippe carralot, 1 taekyu kim, boris lenseigne,2 annette s. In vitro screening and transfection concentration optimization of. Sirnas are strong genesilencing agents that function in a target sequencespecific manner. Lipofectamine 2000 reagent thermo fisher scientific. Effect of cell density on total reporter gene activity. Improved delivery of cas9 proteingrna complexes using. It proves that the sirna targeting the gcacttagcctctatccat of i. Several cell and application specific dnain transfection reagents also have. At both 10 nm and 1 nm p53 sirna, lipofectamine rnaimax transfection reagent provides more effective knockdown relative to nontransfected cells than other rnai reagents, including silentfect biorad, dharmafect dharmacon, and hiperfect qiagen reagents.
Myocytes are long tubular cells that arise from myoblasts and make up muscle fibers in human beings. Solidphase reverse transfection for intracellular delivery of. Download the product protocol from thermo fisher scientific for lipofectamine rnaimax transfection reagent below. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Lipofectamine 2000, 3000, messengermax, and rnaimax.
However, all the transfection reagents tested induced an ifn response in the absence of sirna, which could be minimized by reducing the transfection reagent incubation period. Lipofectamine rnaimax, a new transfection reagent, has been confirmed high efficiency in delivering small interfering rna sirna into rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. Smallsized, stable lipid nanoparticle for the efficient. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. Boese,3 peter sommer,3 auguste genovesio,2 and priscille brodin1 rnai using sirna is a very powerful tool for functional genomics to identify new drug targets and biological pathways. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine rnaimax reagent thermo fisher scientific in. Lipofectamine rnaimax reagent invitrogen was added and the mixture was incubated for 20 min at room temperature to obtain sirna lipofectamine rnaimax complexes. Aug 20, 2015 plasmid dna and mrna were delivered into hek293 cells by lipofectamine 3000, whereas cas9 rnps were delivered with rnaimax. Then, cells were transfected with the candidate valid i.
At both 10 nm and 1 nm p53 sirna, lipofectamine rnaimax transfection reagent provides more effective knockdown relative to nontransfected cells than other rnai reagents, including silentfect biorad, dharmafect dharmacon, and hiperfect qiagen. Aug 11, 2017 cells were transfected with dsdna of indicated lengths and concentrations using lipofectamine 2000, lipofectamine ltx, or lipofectamine rnaimax invitrogen according to the manufacturers instruction with 30. Transfection was carried out as described previously, using lipofectamine rnaimax at a concentration of 0. Reverse transfection is possible but it is recommended to use the 2.
Lipofectamine rnaimax 41% silencing and dharmafect 52% silencing, figure 1. Invitrogen lipofectamine rnaimax transfection reagent. Lipofectamine 2000 was used unless indicated otherwise. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. L 5 5 incubate incubate for 5 minutes at room temperature. Pdf improved delivery of cas9 proteingrna complexes using. Rnai transfection protocols thermo fisher scientific in. Select download format lipofectamine rnaimax reagent protocol. Disregarded effect of biological fluids in sirna delivery. Strong expression of egfp was observed in hek293 cells, mouse. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene. In this study, five commonly used transfection reagents, including lipofectamine 3000, lipofectamine 2000, fugene, rnaimax and. Advanced transfection with lipofectamine 2000 reagent. Nov 28, 2016 the ysk12mend, rnaimax and rnaimax without sirna were incubated in the optimem i media under the same conditions as were used for sirna transfection 30 nm sirna dose for 0.
Lipofectamine rnaimax is used only for small rnas, such as sirnas. Dilute 6 pmol rnai duplex in 50 l optimem i reduced serum medium without serum. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposomebased lipofectamine rnaimax and the hepg2specific, polymerbased genmute, in two cellular models of human hepatoma. Lipofectamine 2000, lipofectamine rnaimax and dharmafect 3, were found to consistently give the best results. Lipofectamine transfection reagents shipped at ambient.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Subcloning is the targeted transfer of a specific dna sequence from the parent vector to the target one. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Plate cells so they will be 7090% confluent at the time of transfection. The location of the break is within the target sequence 3 bases from the ngg pam protospacer adjacent motif 1. According to our results, lipofectamine 3000, fugene and rnaimax showed high transfection efficacy, however, rnaimax may be a better option for majority of cells when lower toxicity is desired. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. The importance of rtqpcr primer design for the detection of. These metrics are regularly updated to reflect usage leading up to the last few days. I did get the knockdown to work when using my sirna with lipofectamine 2000, however. An efficient sirna transfection reagent in human embryonic stem cells rna interference. Efficient gene silencing in hepg2 cells using genmute.
Transfection amounts per well use 10 nm mirna duplex as a starting point. Mix lipofectamine rnaimax gently before use, then dilute 1 l in 50 l optimem i reduced serum medium. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Next, 5 ml of cell suspension including 2 9 106 cells in complete growth medium without antibiotics was added into the cell culture dish. General procedure for transfection of mammalian cells with. Make dna lipofectamine 3000 complexes in serumfree medium such as.
Discussion genmute sirna transfection reagent was developed from. If the volume of lipofectamine rnaimax is too small to dispense accurately, and you cannot pool dilutions, predilute lipofectamine rnaimax 10fold in optimem i reduced serum medium, and dispense a 10fold higher amount should be at least 1. Although sirnas might one day be used in therapy for intractable diseases such as cancers, a number of problems with sirnas must first be overcome. Lipofectamine 2000 has a broad range in nucleic acid. Demonstration of hepatocytetargeted sirna transfection. The functional knockdown was more pronounced when sirna was transfected on day 14 up to 53%, by promofectinhepatocyte at 96hrs than on day 7 20%. After overnight incubation, transfection medium was replaced with rpmi.
Knockdown of target genes in breast cancer cell lines was achieved using scramble andor genespecific sirna ontarget smartpool, thermo scientific and lipofectamine rnaimax. Transfection of cas9 rnp ribonucleoprotein into adherent cells. Lipofectamine rnaimax delivered sirna into both hepatocytes and fibroblasts in hepatopac cocultures. Article views are the countercompliant sum of full text article downloads since november 2008 both pdf and html across all institutions and individuals. May 18, 2016 27 toxicity present 0 10 20 30 40 50 60 lipofectamine rnaimax lipid a lipid b lipid c lipid d lipid e lipid f lipid g 0. Transfection at a later time day 14 generated greater knockdown up. Highefficiency transfection of small rnas and plasmid dna in.
Stealth rnai and sirna transfection concentrations the transfection concentration of a stealth rnai or sirna duplex is determined by dividing the number of moles of sirna used by the final volume of the transfection i. Apr 25, 2011 background sirna technology is a promising tool for gene therapy of vascular disease. Can anyone recommend a protocol for transfection of hepg2. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. See page 2 to view guidelines for transfecting mirnas using lipofectamine rnaimax reagent. Demonstration of hepatocytetargeted sirna transfection and. Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo.
May 25, 2011 the sirnas and lipofectamine rnaimax were from invitrogen. Pulses to use cookies to product availability are subject to consistently transfect many cell transfection of reagent. Add tube b to tube a and incubate at room temperature for five minutes. Scaling up or down lipofectamine 3000 reagent transfections. Feb 18, 2016 identification of lipofectamine crisprmax. In this study, we used three transfection reagents. Incubate 5 min at room temp then mix lipofectamine and sirna dilutions. C2c12 transfection reagent mouse myoblast cells invivo. Reverse transfection an overview sciencedirect topics.
Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with. Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2. Rna interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration while reducing cytotoxicity in your experimental system. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Efficient gene silencing in hepg2 cells using genmute sirna. Lipofectamine 2000 and lipofectamine 3000 reagents deliver dna or cotransfect with excellent transfection performance for protein expression, gene silencing, and functional assays. Combine the diluted rnai duplex with the diluted lipofectamine rnaimax. Transfection of cas9 rnp ribonucleoprotein into adherent. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Two days after transfection with cells at 6080% confluence. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Enhanced green fluorescent protein egfp was clearly seen in eppendorf tubes from harvested cells using lipofectamine 3000 without using a microscope and uv activation. In vivo delivery of sirna targeting vasohibin2 decreases.
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