Prior to transfection, hek 293 cells are preincubated with serum free dmem medium for 1 h. 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. Invitrogen lipofectamine rnaimax transfection reagent provides highest transfection efficiencies on widest variety of cell types for sirnamediated gene knockdown experiments manufacturer. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Alternatively, assays using reporter genes including gfp and luciferease, which are faster. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene.
Cationic liposomes are made of both cationic lipid and neutral phospholipid, conferring positively charged submicron particles, while plasmid dna is a hydrophilic molecule with a highly negative surface charge due to the presence of phosphate groups on. Ribonuclease t 1 is a low molecular weight, highly specific, monomeric. Andersen university of aarhus, department of molecular biology c. Invitrogen lipofectamine rnaimax transfection reagent 0. Invitrogen lipofectamine rnaimax transfection reagent 778075 print share invitrogen lipofectamine rnaimax transfection reagent. Plasmid transfection and expression in p388 cells angelo mancino, birgitta r. Small interfering rna sirna highthroughput screening has a variety of. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas e. With its ability to transfect a broad range of cell types, lipofectamine rnaimax reagent, combined with the invitrogen blockit alexa fluor red fluorescent control, gives you the most versatile approach to all of your knockdown experiments table 1, figure 3.
Jun 01, 2000 cationic liposomemediated transfection lipofection has been used as an efficient method for direct gene transfer both in vitro and in vivo. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Please include all samples to be tested in a single request. However, when the times come to verify the efficiency of the silencing, those cells didnt show any decrease of their rna expression. See page 2 to view a typical rnaimax transfection procedure. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Optimization of sirna transfection for breast and glioma. Mirus considers sample requests for up to three different transfection products and one electroporation product per laboratory. Lipofectamine rnaimax reagent thermo fisher scientific.
For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. Electroporation by nucleofector is the best nonviral. Coronary artery coa and aortic ao smc and ec were transfected with a reporter plasmid, encoding chloramphenicol acetyltransferase type 1 cat, with seven different transfection reagents, two electroporation methods and a photochemical. Is it better to use the forward or reverse transfection. Lipofectamine 2000 transfection reagent dartmouth college. By the way, if you want to try our reagents, do not hesitate to ask me, i can provide test samples for free. Optimization of the transfection condition generally, transfection optimization could be achieved by transfecting cells with sirnas targeting endogenous genes such as lamin ac and gapdh and then analyzing their expression by rtpcr or western blotting. Lipofectamine rnaimax offers a simple protocolno need to remove transfection complexes, or change or add medium following transfection. Adobe acrobat reader dc downloaden gratis pdfviewer voor. I have successfully done this knockdown many times in the past, but i havent been able to get it to work recently. Cas9 nuclease may be used in vivo to create targeted genome modifications. Provides highest transfection efficiencies on widest variety of cell types for sirna. Optimization of sirna transfection for breast and glioma cancer cell cultures rna silencing technology and methods based thereupon are important in todays medical biotechnology. Request a sample of mirus, transitlt1 and transit2020.
Download free adobe acrobat reader dc software for your windows, mac os and android devices to view, print, and comment on pdf documents. To assess transfection efficiency, we recommend using a kif11 stealth select rnai, as. Zhao m1, yang h, jiang x, zhou w, zhu b, zeng y, yao k, ren c. Can anyone recommend a protocol for transfection of hepg2. Transfection of cas9 rnp ribonucleoprotein into adherent. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Cationic liposomes are made of both cationic lipid and neutral phospholipid, conferring positively charged submicron particles, while plasmid dna is a hydrophilic molecule with a highly negative surface charge due to the presence of phosphate groups. I will be using lipofectamine 3000 for plasmid transfection in hepg2 cells. Im trying to express my proteins in nih3t3s, which ive heard are supposedly easy to transfect. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 reagent thermo fisher scientific.
Rnaimax complexes must rnalipofectamine be made in serum free medium such as optimem. 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. According to our results, lipofectamine 3000, fugene and rnaimax. Lipofectamine 2000 and lipofectamine rnaimax transfection. The induction of protein synthesis by exogenous delivery of coding synthetic mrna in desired cells is an auspicious strategy in the fields of basic cell biology, regenerative medicine, treatment of diseases, and reprogramming of cells. Dmriec reagent, lipofectamine 2000, optimem i reduced serummedium. Cationic liposome and plasmid dna complexes formed in serum. There are several ways in which to introduce cas9guide rna complexes into cells. Cationic liposomemediated transfection lipofection has been used as an efficient method for direct gene transfer both in vitro and in vivo. How much lipofectamine rnaimax shoiuld be used for sirna. Ive heard people claim some very high transfection efficiencies using lipofectamine 2000 or brand x lipid reagent.
Rnalipofectamine rnaimax complexes must be made in serum free medium such as optimem. Amounts of reagent and sirna can be scaled up or down depending on the number of cells that will be transfected. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Hek 293 cells are efficiently transfected with lipofectamine, with typically 70% of cells showing fluorescence following transfection with green fluorescent protein gfp. Electroporation by nucleofector is the best nonviral transfection technique in human endothelial and smooth muscle cells.
I also tried lipofectamine rnaimax reagent with epithelial cells and observed a. I am thinking about the cell density or the lipofectamine rnaimax amount that i should use. Mar 03, 2014 the induction of protein synthesis by exogenous delivery of coding synthetic mrna in desired cells is an auspicious strategy in the fields of basic cell biology, regenerative medicine, treatment of diseases, and reprogramming of cells. What is a typical transfection efficiency in % for any of you using lipid based reagents and what cell lines did you use. Ribonuclease t 1 is a low molecular weight, highly specific, monomeric endoribonuclease. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Do you have experience using an efficient transfection reagent for mimics mirna better than lipofectamine. Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2.
Invitrogen lipofectamine rnaimax transfection reagent. Rnaimax complexes must rnalipofectamine be made in serum free medium such as. To place a sample request for mirus bio transfection reagents, please make a selection from the three options below. Selection of transfection reagent is sometimes a puzzling issue.
Sirna question using rnaimax do you change media day after. Lipofectamine 2000 cd is a 100% synthetic version of lipofectamine 2000. Cationic liposome and plasmid dna complexes formed in. Lipofectamine reagents go online to view related products. Ive been having trouble getting efficient sirna knockdown of my protein of interest using lipofectamine rnaimax. L volume of diluted dharmafect transfection reagent in serumfree medium. Cells were transfected with lipofectamine rnaimax complexed with silencer select sirna at 30 nmwell. Invitrogen lipofectamine 3000 transfection reagent 0. For additional information on scaling your transfection reaction, see page 4. Sometimes companies give away free samples if you ask. Lipofectaminea rnaimax reagent thermo fisher scientific. Depending on the cell line and cell density the dharmafect. Report lipofectaminea rnaimax reagent thermo fisher scientific your name.
Lipofectamine rnaimax is proprietary animal origin free, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. These results demonstrated that lipofectamine rnaimax is an efficient and excellent transfection reagent for delivering sirna into hes cells. Invitrogen lipofectamine rnaimax transfection reagent 1. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. Comparison between lipofectamine rnaimax and genmute.
Frequently labs try stay with their traditional technology and do not want to change it due to optimisation issue. This is necessary because serum is replete with rnases that degrade ribozymes. Request a free sample of low toxicity transfection reagents. Low toxicity transfection balance expressiontoxicity toxicity and stress response low tox across cell types request a sample.
Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. Lipofectamine rnaimax troubleshooting ive been having trouble getting efficient sirna knockdown of my protein of interest using lipofectamine rnaimax. Both the reagent and the cy3sirna at different concentrations were diluted 1. Do you have experience using an efficient transfection. Tube a contains optimem media and rnaimax reagent and tube b contains optimem media and sirna. Briefly centrifuge tubes or multiwell plates containing sirna to ensure that the solution is collected at the bottom of the tube. Test serumfree media for compatibility with lipofectamine rnaimax. 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 us. The protocal is from thermo fisher scientific important guidelines for transfection 1. The aim of this study was to determine the optimal nonviral transfection method for use in human smooth muscle cells smc and endothelial cells ec. Our lab routinely works with primary human fibroblasts which are notoriously difficult to transfect, and we require a method that is nontoxic, reliable, and reproducible.
Enzymatic reaction image will open in a new window. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration while reducing cytotoxicity in your experimental system. Lipofectamine 2000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. Shipping conditions back to table of content this kit is shipped on wet ice. Silencer select sirna transfection with lipofectamine rnaimax. Hello there, i purchased 5 sirna sequences from sigma and tried to transfect hek cells using lipofectamine.
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