This work reports for the first time the transfection of this cell line with lipofectamine rnaimax, and with the two types of viromer. Lower volume of jetoptimus is required compared to lipofectamine 3000 in 24well plate excellent cell viability and morphology. Has anyone tried sirna transfection with lipofectamine3000. In this study, we used three transfection reagents lipofectamine rnaimax, oligofectamine and lipofectamine 2000 to deliver sirna into human embryonic stem hes cells and compared the silencing efficiency of enhanced green fluorescent protein transgene and oct4, a key regulator of pluripotency. Add the rnai duplexlipofectamine rnaimax complexes to each well containing cells. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery skip to main content thank you for visiting. This protocol is optimized for transfection in a 6well in vitro culture plate format to perform transfection in different cell culture formats, refer to table2 and invitrogen lipofectamine rnaimax protocol.
Lipofectamine rnaimax invitrogen according to the manufacturers instructions. Vana mimics and inhibitorsnovel tools for mirna functional analysis. Invitrogen lipofectamine rnaimax transfection reagent. 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. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Before you combine the files into one pdf file, use merge pdf to draganddrop pages to reorder or to delete them as you like. Add 6l of lipofectamine2000 from biostores and 100 l of. The cationic lipid c14 is a highly efficient sirna carrier that could be used for the development of new formulations for sirna delivery into cancer cells. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine rnai max, lipofectamine ltx, and glucosefree dmem were from. Lipofectamine rnaimax transfection reagent life technologies according to the manufacturers instructions. Lipofectamine rnaimax transfection reagent provides the highest. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. Invitrogen lipofectamine rnaimax transfection reagent 1.
Interferin sirna mirna transfection reagent polyplus. Lipofectamine rnaimax troubleshooting ive been having trouble getting efficient sirna knockdown of my protein of interest using lipofectamine rnaimax. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and mrna into a wide range of eukaryotic cells. Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Small interfering rna sirna highthroughput screening has a variety of. Tube a contains optimem media and rnaimax reagent and tube b contains optimem media and sirna.
These results demonstrated that lipofectamine rnaimax is an efficient and excellent transfection reagent for delivering sirna into hes cells. Incubate at room temperature for 20 minutes to allow transfection complexes to form. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. A 30% decrease in cell density observed with rnaimax at 25% the recommended concentration compared to ndls and controls. I tried those sequences on other cell line and they dont have any affect on the expression level of the targeted gene. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection of crisprcas9 nuclease nls ribonucleoprotein. Lipofectamine 2000 and lipofectamine rnaimax transfection.
Scaling up or down transfections to transfect cells in different tissue culture formats, vary the amounts of lipofectamine rnaimax, rnai duplex, cells, and medium used in proportion to the relative surface area, as shown in the table. Invitrogen lipofectamine 3000 transfection reagent. Ab 7900 48h eg5 sirna 48h cdc sirna 8200 cellular detection system mrna knockdown 18s neg1 surv protein knockdown gapdh survivin biology if 0 2000 3000 4000 5000 6000 r nc r 23 r nc r 21 r nc r 15a r nc r 16 r nc r 19a no r u duallight reagent functional analysis using sirna. Lipofectamine rnaimax, what medium for transfection. The transfection is designed for one rna amount combined with one amount of lipofectamine rnaimax. High throughput rnai assay optimization using adherent. Use this procedure to reverse transfect stealth rnai or sirna into mammalian cells in a 24well format for other formats, see. Plate cells so they will be 7090% confluent at the time of transfection.
Improved delivery of cas9 protein grna complexes using. Due to the multitude of reagents and cell types, rnai experiment optimization can be timeconsuming. Chengqi yis lab from peking university and authenticated by china center for type culture collection wuhan, china as free from. Improved delivery of cas9 proteingrna complexes using. Ab 7900 48h eg5 sirna 48h cdc sirna 8200 cellular detection system mrna knockdown 18s neg1 surv protein knockdown. May 11, 2016 the intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery skip to main content thank you for visiting. Mit center for biomedical innovations biomanufacturing. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. Shop online for a wide selection of invitrogen lipofectamine rnaimax transfection reagent provides highest transfection efficiencies on widest variety of cell types for. Transfection of cas9 rnp ribonucleoprotein into adherent. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. I have successfully done this knockdown many times in the past, but i havent been able to get it to work recently. Reaching high transfection efficiencies is often at the expense of cellular integrity, with adverse effects on cell cycle, metabolism and signaling pathways. Lipofectamine 2000 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind.
The prepared mix is enough to have triplicates 96well, duplicates 24well, and single well 6well transfections, and account for pipetting variations. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectaminea rnaimax reagent thermo fisher scientific. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration while reducing cytotoxicity in your experimental system. Pool dishes of cells that have been prepared for transfection. Lipofectamine 2000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. Rnaimax reverse transfections lipofectamine thermo fisher. There are several ways in which to introduce cas9guide rna complexes into cells.
Aosmc were seeded at a density of 30008000 cellswell of a 96well plate. This protocol is optimized for transfection in a 6wellin vitroculture plate format. Add tube b to tube a and incubate at room temperature for five minutes. See page 2 to view a typical rnaimax transfection procedure. Lipofectamine reagents go online to view related products. It increases the likelihood of successful cleavage and recombination with talens or crisprs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes. No other transfection reagent for rna interference rnai experiments provides such easy and ef. Lipofectamine rnaimax invitrogen was used in the amount of. Optimization of sirna transfection for breast and glioma cancer cell cultures rna silencing technology and methods based thereupon are important in todays medical biotechnology. Use this procedure to forward transfect sirna into mammalian cells in a 24well format for other formats, see scaling up or down transfections, page 3. The intracellular trafficking mechanism of lipofectamine.
Transfection and genome engineering fisher scientific. Lipofectamine reagents rnaimax transfection reagent is a lipofectamine proprietary formulation for transfecting small rnas e. In the present study, several parameters were tested to increase silencing efficacy in raw 264. Amounts of reagent and sirna can be scaled up or down depending on the number of cells that will be transfected. In this study adherent cell cytometry was used to rapidly optimize sirna transfection in human aortic vascular smooth muscle cells aosmc. From our experiences, we noticed that lipofectamine rnaimax is superior to lipofectamine 2000 to the extent of cytotoxicity and rnai efficiency, and that cytotoxicity of lipofectamine 2000 varies lot by lot. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine rnaimax reagent thermo fisher scientific us. Nucleic acidlipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serumantibiotic. The corresponding values for lipofectamine rnaimax rnaimax. A 30% decrease in cell density observed with rnaimax at 25% the. Original article enhanced specificity of hpv16 e6e7 sirna by rnadna chimera modification k yamato1, n egawa2, s endo3, k uitei4, t yamada5, k saigo4, i hyodo3, t kiyono2 and i nakagawa1 1section of bacterial pathogenesis, graduate school of medical and dental science, tokyo medical and dental university, tokyo, japan. However, the transfection of sirna to human immune cells is difficult. The cells were incubated at 37c for 3 days before the assay.
Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2. This gives a final volume of 600 l and a final rna concentration of 10 nm. Smallsized, stable lipid nanoparticle for the efficient delivery of. To achieve the maximal rnai activity and lowest cytotoxicity, optimization of lipofectamine 2000. Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. For additional information on scaling your transfection reaction, see page 4. The goal of the project was to determine if the transcription factor nfia mediates the cytotoxicity of a novel anticancer therapeutic.
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 with this agent was functional, evidenced by the decreased expression of protein kinase 9 ptk9, used as a positive control. Incubate the cells 2448 hours at 37c in a co2 incubator until you are ready to assay for gene knockdown. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem. The single guide rna sgrna vector targeting gfp was obtained from dr. Easy optimization coupled with simple, cell linespecific protocols. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Qc specifications back to table of content lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Interferin is the reagent of choice for delivering mirna, mirna mimics or premirnas.
To change the order of your pdfs, drag and drop the files as you want. Quotation no p2608185 life technologies corporation. Antisense oligonucleotides bind and inhibit mirnas. Feb 18, 2016 lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of cell lines. I would like to use lipofectamine rnaimax for transfection of cows luteal cell with sirna duplex in 24 well plates. Lipofectamine 3000 reagent is suitable for novel genome editing applications. However, when the times come to verify the efficiency of the silencing, those cells didnt show any decrease of their rna expression. Thee two most important factors in choosing transfecting reagent are. Quantitative realtime pcr showed approximately 90% knockdown of oct4 transcript with cognate oct4 sirna transfection by lipofectamine rnaimax compared to control sirna. Lipofectamine rnaimax reagent thermo fisher scientific. Lipofectamine dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Optimization of sirna transfection for breast and glioma. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. I would like to ask about the medium in which the transfection should be.
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 rnaimax transfection reagent lipofectamine rnaimax reagent offers an advanced, ef. Lipofectamine rnaimax decreases cho cell growth by 30% relative to ndls and untreated controls. Highly efficient cationic ethylphosphatidylcholine sirna carrier for. Steinauer pnas supplementary information 103018 tracking. All uploads and downloads are deemed secure and files are permanently deleted from the smallpdf servers within an hour. Dilute sirna duplex in 250 ul growth medium or optimem i reduced serum.
Enhanced specificity of hpv16 e6e7 sirna by rnadna. Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. However, delivery of the cas9 rnps by neon electroporation was effective across all different cell lines tested and often was the only method that could produce indels. Lipofectamine 2000 transfection reagent from invitrogen. Add solution from tube 1 rnp complex to diluted lipofectamine rnaimax in tube 2 and mix well by pipetting up and down. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid. In addition, method adaptation to the hts format was. Rnaimax has unmatched performance, delivering greater knockdown with less sirna. Validation of a transfection protocol to study the. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 protein grna complexes into a variety of cell lines. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
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