This products resides on a fisher scientific gsa or va contract. Zhao m1, yang h, jiang x, zhou w, zhu b, zeng y, yao k, ren c. Scaling up or down lipofectamine 3000 reagent transfections. What is composition of lipofectamine rnaimax reagent. 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. As nehad pointed out, you can do reverse transfection with lipofectamine 2000 or rnaimax. In this study, five commonly used transfection reagents, including lipofectamine 3000, lipofectamine 2000, fugene, rnaimax and. Lipofectamine rnaimax transfection reagent lipofectamine rnaimax reagent offers an advanced, ef. Establishment of lipofection protocol for efficient mir21. Reverse transfection is possible but it is recommended to use the 2. Provides highest transfection efficiencies on widest variety of cell types for sirna.
Quantitative realtime pcr showed approximately 90% knockdown of oct4 transcript with cognate oct4 sirna transfection by lipofectamine rnaimax compared to control sirna. The intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery. Invitrogen lipofectamine rnaimax transfection reagent 1. Shop online for a wide selection of invitrogen lipofectamine rnaimax transfection reagent provides highest transfection efficiencies on widest variety of cell types for. Lipofectamine rnaimax reagent thermo fisher scientific us. Lipofectamine rnaimax transfection reagent from thermo fisher. 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.
Mix lipofectamine rnaimax gently before use, then add 1 l lipofectamine rnaimax to each well containing the diluted rnai molecules. Invitrogen lipofectamine rnaimax transfection reagent. Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2. Transfection and genome engineering fisher scientific. Comparison of small interfering rna sirna delivery into. Timeline steps procedure details day 0 1 seed cells to be 60. Incubate 5 min at room temp then mix lipofectamine and sirna dilutions. Amounts of reagent and sirna can be scaled up or down depending on the number of cells that will be transfected. 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. If you are viewing this page as a nonregistered user, the prices displayed is list price. I would like to use lipofectamine rnaimax for transfection of cows luteal cell with sirna duplex in 24 well plates. Dilute lipofectamine rnaimax reagent in optimem medium 2. As a result, sirna can be delivered into hes cells more efficiently by lipofectamine rnaimax compared with the other two transfection reagents and high efficient knockdown of target genes was.
Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. No other transfection reagent for rna interference rnai experiments provides such easy and ef. Lipofectamine 2000, lipofectamine rnaimax and dharmafect 3, were found to consistently give the best results. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The intracellular trafficking mechanism of lipofectamine.
Lipofectamine is a cationic liposome formulation, which complexes with negatively charged nucleic acid molecules to allow them to overcome the electrostatic repulsion of the cell membrane. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. Advanced transfection with lipofectamine 2000 reagent. I have successfully done this knockdown many times in the past, but i havent been able to get it to work recently. Lipofectamine rnaimax invitrogen by life technology 778075.
Lipofectamine 3000 and lipofectamine rnaimax served as controls. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. 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. Lipofectamine rnaimax invitrogen was used in the amount of. The advantages of lipidbased transfection reagents have permitted their widespread use in molecular biology and gene therapy. Sw1463 3d tumour spheroids we used a fluorescent cy3conjugated nontargeting control sirna and formed complexes with lipofectamine rnaimax in either optimem, or.
I use 24well plates for transfections in triplicates. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna. 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. Thee two most important factors in choosing transfecting reagent are. Improved delivery of cas9 proteingrna complexes using. Rnaimax reverse transfections lipofectamine thermo. This protocol isintended for the transfection of adherent mammalian tissue culture cells. No other transfection reagent for rna interference rnai experiments provides such easy and. I would like to ask about the medium in which the transfection should be. Lipidbased transfection reagents exhibit cryoinduced. Invitrogen lipofectamine rnaimax transfection reagent 778150 print share invitrogen lipofectamine rnaimax transfection reagent. Lipofectamine reagents go online to view related products.
Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. 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. Lipofectamine 2000 and lipofectamine rnaimax transfection. Reverse transfection an overview sciencedirect topics. Lipofectamine 2000 and lipofectamine 3000 reagents deliver dna or cotransfect with excellent transfection performance for protein expression, gene silencing, and functional assays. This procedure is designed for sirna in combination with lipofectamine rnaimax.
Lipofectamine rnaimax, what medium for transfection. Even though cell lines differ in their experimental properties, this protocol will yield efficient transfection at low nano molar concentrations in most of the commonly. Imlf can be transfected using lipofectamine 3000 thermo fisher scientific, waltham, ma, usa but transfection efficiency is higher using recombinant adenovirus. Lipofectaminea rnaimax reagent thermo fisher scientific. Combine the diluted sirna duplex with the diluted lipofectamine rnaimax. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Invitrogen lipofectamine rnaimax transfection reagent 778075 print. Invitrogen lipofectamine rnaimax transfection reagent 0. I did get the knockdown to work when using my sirna with lipofectamine 2000, however. Page 4 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. 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.
Optimized delivery of sirna into 3d tumor spheroid cultures. What amount of lipofectamine rnaimax and sirna concentration. These results demonstrated that lipofectamine rnaimax is an efficient and excellent transfection reagent for delivering sirna into hes cells. See page 2 to view a typical rnaimax transfection procedure. This study outlines the effect of cryomanipulation of a cationic lipidbased formulation, lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without. What amount of lipofectamine rnaimax and sirna concentration should i use to transfect primary culture of sertoli cells in a six well plate. An efficient sirna transfection reagent in human embryonic stem cells rna interference. Lipofectamine 2000, 3000, messengermax, and rnaimax. Can anyone recommend a protocol for transfection of hepg2.
For each well to be transfected, prepare rnai duplexlipofectamine rnaimax complexes as follows. Lipofectamine rnaimax rnaimax, a new cationic lipofection reagent, has shown higher efficiency in delivering mirna into many stem cells and cell lines. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Dilute 6 pmol rnai duplex in 100 l optimem i medium without serum in the well of the tissue culture plate. Mix lipofectamine rnaimax gently before use, then dilute 3. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high. The lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna, and lipofectamine messengermax reagent delivers mrna. Lipofectamine rnaimax transfection reagent delivery of sirna. Gfp plasmid to sirna while doing lipofectamine 2000 transfection. Plate cells in a 6well culture dish 1 day before transfection. See the engen sgrna synthesis kit manual for further details. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using lipofectamine. Lipofectamine 2000 transfection reagent from invitrogen.
Tube a contains optimem media and rnaimax reagent and tube b contains optimem media and sirna. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types. Plate cells so they will be 7090% confluent at the time of transfection. The prepared mix is sufficient for triplicate transfections with overage. 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. The cas9 rnps in optimem medium were added to the transfection. Make dna lipofectamine 3000 complexes in serumfree medium such as. Lipofectamine rnaimax is used only for small rnas, such as sirnas. 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 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. Cells should reach 5070% confluency at time of transfection. Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using tat or ccr5specific small interfering rna s. Mix lipofectamine rnaimax gently before use, then add 1 l lipofectamine rnaimax to each. Lipofectamine rnaimax troubleshooting ive been having trouble getting efficient sirna knockdown of my protein of interest using lipofectamine rnaimax.
Lipofectamine rnaimax transfection reagent from thermo. 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. Mix and incubate for 20 minutes at room temperature. Invitrogen lipofectamine rnaimax transfection reagent the gold standard in delivery of sirna and mirna for gene knockdown experiments effectiveprovides maximum knockdown with less sirna and low cytotoxicity versatileworks across a wide range of cell types sirna delivery invitrogen crisprmax cas9 transfection reagent. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid. Invitrogen lipofectamine 3000 transfection reagent. Smallsized, stable lipid nanoparticle for the efficient. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Rnaimax reverse transfections lipofectamine thermo fisher. Add tube b to tube a and incubate at room temperature for five minutes.
Lipofectamine rnaimax transfection reagent thermofisher. For additional information on scaling your transfection reaction, see page 4. Transfection of cas9 rnp ribonucleoprotein into adherent cells. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration while reducing cytotoxicity in your experimental system. Lipofectamine rnaimax reagent thermo fisher scientific.
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