Red lightactivated sirna therapy rossensitive crosslinked. Efficient and specific knockdown of small noncoding rnas. Plate cells so they will be 7090% confluent at the time of transfection. 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. Tube a contains optimem media and rnaimax reagent and tube b contains optimem media and sirna. Invitrogen lipofectamine rnaimax transfection reagent 1. Lipofectamine 2000 reagent thermo fisher scientific. Comparison of small interfering rna sirna delivery into. If you are viewing this page as a nonregistered user, the prices displayed is list price. Quantitative realtime pcr showed approximately 90% knockdown of oct4 transcript with cognate oct4 sirna transfection by lipofectamine rnaimax compared to control sirna. 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. The latter can lead to false positive results when realtime rtqpcr alone is used to measure target mrna knockdown. 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. It is not necessary to remove complexes or changeadd. These results demonstrated that lipofectamine rnaimax is an efficient and excellent transfection reagent for delivering sirna into hes cells. 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. Refer to the cell lines database for a list of cell types successfully transfected. The intracellular trafficking mechanism of lipofectamine.
Apr 25, 2011 sirna technology is a promising tool for gene therapy of vascular disease. Lipofectamine rnaimax transfection reagent from thermo. Zhao m1, yang h, jiang x, zhou w, zhu b, zeng y, yao k, ren c. Mix and incubate for 20 minutes at room temperature. Improved delivery of cas9 proteingrna complexes using. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture medium, in the. Rnaimax reverse transfections lipofectamine thermo fisher. Transfection amounts lipofectamine reagents rnaimax transfection reagent is a lipofectamine proprietary formulation for transfecting small rnas e. Combine the diluted sirna duplex with the diluted lipofectamine rnaimax. 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. Lipofectamine rnaimax transfection reagent from thermo fisher.
There are several ways in which to introduce cas9guide rna complexes into cells. Instructional calendar 20162017 greene central school district. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Aosmc were seeded at a density of 30008000 cellswell of a 96well plate. Fluorescently labeled famsirna, cy5sirna, the black hole quencher3 bhq3, a dark quencher. Lipofectamine rnaimax lipo was used as a positive control. Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. Cells were stained for pluripotency with the ssea4 antibody, visualized by. Lipofectamine 2000, lipofectamine rnaimax and dharmafect 3, were found to consistently give the best results. Transfecting sirna using lipofectamine rnaimax pin. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Mix lipofectamine rnaimax gently before use, then dilute 3. 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. For adaptations of the basic protocol below, follow.
Lipofectamine 2000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Lipofectamine reagents go online to view related products. Add tube b to tube a and incubate at room temperature for five minutes. I would like to use lipofectamine rnaimax for transfection of cows luteal cell with sirna duplex in 24 well plates.
Lipofectamine rnaimax reagent thermo fisher scientific us. The concentration of sirna is 10nmole working 50micromolar. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Has anyone tried sirna transfection with lipofectamine3000. 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. The following cell lines have been successfully transfected with the indicated transfection reagents. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Incubate the cells 2448 hours at 37c in a co2 incubator until you are ready to assay for gene knockdown. 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. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Red lightactivated sirna therapy rossensitive cross. Lipofectamine 3000 protocol written out with timing specifics. Mammary epithelial basal medium mebm and megm singlequots supplements were obtained from lonza.
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. Cas9 nuclease may be used in vivo to create targeted genome modifications. Dendritic peptide bolaamphiphiles for sirna delivery to. Lipofectamine 2000 transfection reagent dartmouth college. 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 reagent thermo fisher scientific. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. Due to the multitude of reagents and cell types, rnai experiment optimization can be timeconsuming. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. The use of rnai to analyse gene function in vitro is now widely applied in biological research. For each well to be transfected, prepare rnai duplexlipofectamine rnaimax complexes as follows.
Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas e. 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. Highest transfection efficiency in many cell types and formats e. Add the rnai duplexlipofectamine rnaimax complexes to each well containing cells. Lipofectamine 2000 procedure for simultaneously transfecting. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. A h9 escs or b ipscs were transfected using lipofectamine 3000 reagent. Lipofectamine rnaimax, what medium for transfection.
Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Lipofectamine rnaimax transfection reagent delivery of sirna. For additional information on scaling your transfection reaction, see page 4. Can anybody give me exact protocol for sirna transfection using. I would like to ask about the medium in which the transfection should be. Out of the five transfection systems tested, fugene and rnaimax demonstrated the highest. You can generally use other serumfree medium instead of optimem. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
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. 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. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types. Mix lipofectamine rnaimax gently before use, then dilute 1 l in 50 l optimem i reduced serum medium. Lipofectaminea rnaimax reagent thermo fisher scientific. Pool dishes of cells that have been prepared for transfection. Dilute 6 pmol rnai duplex in 50 l optimem i reduced serum medium without serum. Invitrogen lipofectamine rnaimax transfection reagent. This products resides on a fisher scientific gsa or va contract. Incubate 5 min at room temp then mix lipofectamine and sirna dilutions. Cells were transfected with lipofectamine rnaimax complexed with silencer select sirna at 30 nmwell. Lipofectamine contains lipid subunits that can form. Despite this, a satisfactory mechanismbased explanation of their. Follow manufacturers handbook protocol unless otherwise indicated.
Superior knockdown with lipofectamine rnaimax transfection reagent compared to competing sirna transfection reagents. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. 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 lipofectamine rnaimax transfection reagent and hoechst 33342 from thermo fisher scientific were used as suggested by the supplier. See page 2 to view guidelines for transfection of sirnas using lipofectamine. Amounts of reagent and sirna can be scaled up or down depending on the number of cells that will be transfected.
Plate cells so they will be 6080% confluent at the time of transfection. 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 2000 and lipofectamine rnaimax transfection. I did get the knockdown to work when using my sirna with lipofectamine 2000, however. 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. Efficient and specific knockdown of small noncoding rnas in. Dilute lipofectamine rnaimax reagent in optimem medium 2 dilute sirna in optimem medium 3 add diluted sirna to diluted lipofectamine rnaimax reagent 1. This gives a final volume of 600 l and a final rna concentration of 10 nm. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and mrna into a wide range of eukaryotic cells. High throughput rnai assay optimization using adherent cell. 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. Transfection of cas9 rnp ribonucleoprotein into adherent.
81 359 1211 241 34 964 824 80 1189 1292 1433 1241 270 423 220 354 1047 1027 899 777 854 513 906 341 1457 273 1487 956 1264 1452 1225 1424 86 1142 418 1332 47 29 907 435 1257