A h9 escs or b ipscs were transfected using lipofectamine 3000 reagent. Refer to the cell lines database for a list of cell types successfully transfected. Transfection of cas9 rnp ribonucleoprotein into adherent. 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. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e.
Lipofectamine contains lipid subunits that can form. Lipofectamine 2000 reagent thermo fisher scientific. Efficient and specific knockdown of small noncoding rnas in. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Incubate the cells 2448 hours at 37c in a co2 incubator until you are ready to assay for gene knockdown. Lipofectamine 2000, lipofectamine rnaimax and dharmafect 3, were found to consistently give the best results. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and mrna into a wide range of eukaryotic cells. 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 contains lipid subunits that can form liposomes in an aqueous environment, which entrap the.
Superior knockdown with lipofectamine rnaimax transfection reagent compared to competing sirna transfection reagents. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture medium, in the. Invitrogen lipofectamine rnaimax transfection reagent. Dilute lipofectamine rnaimax reagent in optimem medium 2 dilute sirna in optimem medium 3 add diluted sirna to diluted lipofectamine rnaimax reagent 1. I would like to ask about the medium in which the transfection should be. Transfection amounts lipofectamine reagents rnaimax transfection reagent is a lipofectamine proprietary formulation for transfecting small rnas e. Amounts of reagent and sirna can be scaled up or down depending on the number of cells that will be transfected. Red lightactivated sirna therapy rossensitive cross. Quantitative realtime pcr showed approximately 90% knockdown of oct4 transcript with cognate oct4 sirna transfection by lipofectamine rnaimax compared to control sirna.
Mix lipofectamine rnaimax gently before use, then dilute 1 l in 50 l optimem i reduced serum medium. Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. You can generally use other serumfree medium instead of optimem. 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. 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 reagent thermo fisher scientific us. I did get the knockdown to work when using my sirna with lipofectamine 2000, however. Instructional calendar 20162017 greene central school district.
Cells were stained for pluripotency with the ssea4 antibody, visualized by. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The cell density per well and amount of lipofectamine rnaimax used per well were as follows. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. The intracellular trafficking mechanism of lipofectamine. If you are viewing this page as a nonregistered user, the prices displayed is list price. The lipofectamine rnaimax transfection reagent and hoechst 33342 from thermo fisher scientific were used as suggested by the supplier. Lipofectamine rnaimax transfection reagent from thermo. Combine the diluted sirna duplex with the diluted lipofectamine rnaimax. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. In this study adherent cell cytometry was used to rapidly optimize sirna transfection in human aortic vascular smooth muscle cells aosmc. Lipofectamine rnaimax transfection reagent delivery of sirna.
For each well to be transfected, prepare rnai duplexlipofectamine rnaimax complexes as follows. Out of the five transfection systems tested, fugene and rnaimax demonstrated the highest. Pool dishes of cells that have been prepared for transfection. Red plasma membrane stain, nucblue live readyprobes reagent, lipofectamine rnaimax transfection reagent and vybrant mtt cell proliferation assay kit were purchased from thermo fisher scientific.
Add the rnai duplexlipofectamine rnaimax complexes to each well containing cells. Dilute 6 pmol rnai duplex in 50 l optimem i reduced serum medium without serum. Despite this, a satisfactory mechanismbased explanation of their. 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 3000 protocol written out with timing specifics. 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.
Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas e. Tube a contains optimem media and rnaimax reagent and tube b contains optimem media and sirna. This products resides on a fisher scientific gsa or va contract. Cells were transfected with lipofectamine rnaimax complexed with silencer select sirna at 30 nmwell. Lipofectamine rnaimax reagent thermo fisher scientific. Invitrogen lipofectamine rnaimax transfection reagent 1. Plate cells so they will be 6080% confluent at the time of transfection. Lipofectamine 2000 procedure for simultaneously transfecting. Incubate 5 min at room temp then mix lipofectamine and sirna dilutions. For additional information on scaling your transfection reaction, see page 4. Invitrogen lipofectamine rnaimax transfection reagent 0.
The following cell lines have been successfully transfected with the indicated transfection reagents. These results demonstrated that lipofectamine rnaimax is an efficient and excellent transfection reagent for delivering sirna into hes cells. Rnaimax reverse transfections lipofectamine thermo fisher. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Mammary epithelial basal medium mebm and megm singlequots supplements were obtained from lonza. Mix and incubate for 20 minutes at room temperature. Lipofectamine rnaimax, what medium for transfection. Can anybody give me exact protocol for sirna transfection using. Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. Has anyone tried sirna transfection with lipofectamine3000.
Lipofectamine rnaimax transfection reagent from thermo fisher. High throughput rnai assay optimization using adherent cell. Comparison of small interfering rna sirna delivery into. Ontarget plus non targeting sirnas d 0018100120, ontarget plus smart pool, human bmpr2 sirnas l005309000010 and ontarget plus smart pool, human brca1 sirnas l00346010 were transfected into s using lipofectamine rnaimax ec. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. It is not necessary to remove complexes or changeadd. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000.
Lipofectamine rnaimax lipo was used as a positive control. Improved delivery of cas9 proteingrna complexes using. 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, several difficulties are associated with its use in vivo, mainly relating to inefficient delivery and nonspecific effects of short rna duplexes in animal models. 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. How much lipofectamine rnaimax shoiuld be used for sirna. This gives a final volume of 600 l and a final rna concentration of 10 nm. The use of rnai to analyse gene function in vitro is now widely applied in biological research.
Lipofectamine 2000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Mix lipofectamine rnaimax gently before use, then dilute 3. Lipofectaminea rnaimax reagent thermo fisher scientific. Apr 25, 2011 sirna technology is a promising tool for gene therapy of vascular disease. Follow manufacturers handbook protocol unless otherwise indicated. 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. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. I will try different conditions of confluency and lipofectamine rnaimax, if it doesnt work, i will try the reagent you.
Systematic screening of commonly used commercial transfection. Fluorescently labeled famsirna, cy5sirna, the black hole quencher3 bhq3, a dark quencher. Add tube b to tube a and incubate at room temperature for five minutes. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. 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 reagents go online to view related products. Lipofectamine 2000 transfection reagent dartmouth college. The latter can lead to false positive results when realtime rtqpcr alone is used to measure target mrna knockdown. Transfecting sirna using lipofectamine rnaimax pin. Aosmc were seeded at a density of 30008000 cellswell of a 96well plate. I would like to transfect 293t cells with lipofectamine 3000. Dendritic peptide bolaamphiphiles for sirna delivery to. Lipofectamine rnaimax is a proprietary formulation specifically developed for the transfection of sirna and stealth rnai duplexes into eukaryotic cells.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Red lightactivated sirna therapy rossensitive crosslinked. Cas9 nuclease may be used in vivo to create targeted genome modifications. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Zhao m1, yang h, jiang x, zhou w, zhu b, zeng y, yao k, ren c.
Due to the multitude of reagents and cell types, rnai experiment optimization can be timeconsuming. Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. See page 2 to view guidelines for transfection of sirnas using lipofectamine. Plate cells so they will be 7090% confluent at the time of transfection. Highest transfection efficiency in many cell types and formats e. There are several ways in which to introduce cas9guide rna complexes into cells. I would like to use lipofectamine rnaimax for transfection of cows luteal cell with sirna duplex in 24 well plates. 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.
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