Lipidbased transfection reagents exhibit cryoinduced increase in. It is possible to plate and transfect the cells on the same day, but cells must adhere to. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Smallsized, stable lipid nanoparticle for the efficient. Combine the diluted sirna duplex with the diluted lipofectamine rnaimax.
Works effectively with all common cell lines as well as with many challenging ones, and can be used in media with or without serum. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Prewarm 50ml of optimum stored in cold room at 4c 10min. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. For additional information on scaling your transfection reaction, see page 4. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments.
May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Whats the difference between lipofectine and lipofectamine. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Improved delivery of cas9 proteingrna complexes using. 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. Invitrogen lipofectamine rnaimax transfection reagent offers an advanced, efficient solution for sirna delivery. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. 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. Amounts of reagent and sirna can be scaled up or down depending on the number of cells that will be transfected. 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. Transfection efficiency in a cancer cell line panel.
Lipofectamine 2000, 3000, messengermax, and rnaimax. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 3000 and lipofectamine rnaimax served as controls. I did get the knockdown to work when using my sirna with lipofectamine 2000, however. By shipping these reagents at ambient temperatures. Comparison of transfection through lipofectamine and polyeth. Maintain p19 cells undifferentiated in mem with 10% serum 7. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines.
At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Incubate 5 min at room temp then mix lipofectamine and sirna dilutions. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. The process associated with cellular uptake played a role in effective gene silencing effect. I would like to use lipofectamine rnaimax for transfection of cows luteal cell with sirna duplex in 24 well plates. As always, use good cell culture practices and wear your personal protective equipment. Optimizing transfections to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rnai. Rnaimax transfection reagentsat ambient rather than cold temperatures. Lipofectamine 2000 transfection protocol 10cm plates reagents. Superior knockdown with lipofectamine rnaimax transfection reagent compared to competing sirna transfection reagents. 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.
The amount of lipofectamine 2000 should be changed in proportion to the culture medium. 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 each well to be transfected, prepare rnai duplexlipofectamine rnaimax complexes as follows. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectaminea rnaimax reagent thermo fisher scientific. Transfecting sirna using lipofectamine rnaimax pin. Lipofectamine transfection rnaimax reagent lipofectamine 2000, 3000 lipofectamine 3000 great results could be better poor results great results could be better poor results try reverse transfection different cell density increased sirna concentration 1 2 3 try different dna concentration different cell density 1 2 try neon transfection system viruses with shrna 1 2 try. I use 24well plates for transfections in triplicates. Rnalipofectamine rnaimax complexes must be made in serumfree medium such as optimem. Lipofectamine 2000 is a highperformance transfection reagent for gene expression and gene silencing.
Tube a contains optimem media and rnaimax reagent and tube b contains optimem media and sirna. Ideal for transient protein expression or highthroughput rnai transfections. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to transfect 17 cell lines with a gfpexpressing plasmid in a 24well plate format using 0. Add tube b to tube a and incubate at room temperature for five minutes. Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. Lipofectamine 2000, 3000, messengermax, and rnaimax reagents are cationiclipid. 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 is used only for small rnas, such as sirnas. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. The intracellular trafficking mechanism of lipofectamine.
So i asked one of my lab mates and he told me to use just 1ul lipofectamine for 20pmol of sirna thats the protocol specificationsbut instead for each well, use that amount for three wells. Hepatocellular carcinoma sixth most common cancer third leading cause of cancer related deaths early stage treatments liver transplantation and surgical resection metastasis and recurrence dna will be transfected into the huh7 cell line ability to grow without serum growth. Life technologies is committed to providing researchers and scientists. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. These results demonstrated that lipofectamine rnaimax is an efficient and. Transfection efficiency and cell toxicityviability, reagent dose response a dose response from. No other sirna specific transfection reagent provides such easy and efficient sirna delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hardtotransfect cell types. 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. Works effectively with all common cell lines as well as with many challenging ones, and can. Highest transfection efficiency in many cell types and formats e. Can anybody give me exact protocol for sirna transfection using. Mix and incubate for 20 minutes at room temperature. Lipofectamine 2000, lipofectamine rnaimax and dharmafect 3, were found to consistently give the best results. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells.
Dilute lipofectamine rnaimax reagent in optimem medium 2 dilute sirna in optimem medium 3 add diluted sirna to diluted lipofectamine rnaimax reagent 1. Add 6l of lipofectamine2000 from biostores and 100 l of. 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. Higher densities can lead to spontaneous differentiation. Mix lipofectamine rnaimax gently before use, then dilute 3. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent and suspension cell lines.
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. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. No 20 is optimized for use with lipofectamine 2000, and is not recommended for lipofectamine rnaimax.
Lipofectamine rnaimax, what medium for transfection. Invitrogen lipofectamine rnaimax transfection reagent. Invitrogen lipofectamine rnaimax transfection reagent 0. Plate cells in a 6well culture dish 1 day before transfection. Transfection and genome engineering fisher scientific. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Lipofectamine rnaimax reagent thermo fisher scientific us. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas e. Product is guaranteed for 6 months from the date of shipment if. To elucidate this, a comparison in plasmid transfection efficiency between lf2000 and other lipidbased lipofectamine rnaimax, dotap. It takes nearly 120 barrels of crude oil equivalents and 150 mwh of power annually to make the 9,000 kg of eps coolers needed to ship these reagents1,2. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. Lipofectamine 2000 and lipofectamine rnaimax transfection.
Invitrogen lipofectamine 2000 transfection reagent fisher. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine contains lipid subunits that can form. Invitrogen lipofectamine 2000 transfection reagent. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Nov 28, 2016 the corresponding values for lipofectamine rnaimax rnaimax were 37%, 56%, 43% and 19%, respectively. 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 transfection reagent from thermo. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types. The corresponding values for lipofectamine rnaimax rnaimax were 37%, 56%, 43% and 19%, respectively. However, significant toxicity from lipofectamine 2000 was also reported. Despite this, a satisfactory mechanismbased explanation of their. 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. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Date 20 july 2012 description lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and.
Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. An efficient sirna transfection reagent in human embryonic stem cells, authorming zhao and hong yang and xingjun jiang and wen zhou and bin zhu and ying zeng. Dilute 6 pmol rnai duplex in 50 l optimem i reduced serum medium without serum. Formulation of lipofectamine 2000 and lipofectin signagen blog. Mix lipofectamine rnaimax gently before use, then dilute 1 l in 50 l optimem i reduced serum medium. Lipofectamine 2000 reagent and lipofectamine ltx reagent. Comparison of small interfering rna sirna delivery into. I would like to ask about the medium in which the transfection should be.
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