Jun 01, 2004 lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. The medium was changed to full dmemh dulbeccos modified eagles medium with high glucose, invitrogen after 4 h. Silencing of gene expression in cultured cells using small. Rnabased, transient modulation of gene expression in human. Lipofectamine rnaimax, a new transfection reagent, has been confirmed high efficiency in delivering small interfering rna sirna into rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. Transfecting sirna into mammalian cells using lipofectaminez 2000. With automated, highthroughput systems, a complexing volume of 50 l is recommended for transfections in 96well plates. To study the effect of sirna knockdown of an endogenous gene, sirna transfection should be done first using an rnai specific. The transfection efficacy of lipofectamine 2000 was compromised by.
Comparison of small interfering rna sirna delivery into bovine. L 30 pmol 4 add diluted sirna to diluted lipofectamine rnaimax reagent 1. Transfect cells at high cell density for high efficiency, high. Add the oligomerlipofectamine 2000 complexes to each well containing cells and medium. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and dna concentrations, liposomedna complexing time, and the presence or absence of media components such as antibiotics and serum. Invitrogen lipofectamine rnaimax transfection reagent. Mix lipofectamine 2000 cd reagent gently before use, then dilute 0. Lipofectamine 2000 lipo2000 delivery system is commonly used for short interfering rna sirna transfection, whereas the cellular responses have attracted little attention. Optimem is the choice of medium for lipofectamine rnaimax and other lipofectamine reagents eg lipofectamine 2000. The delivery of sirnas with chemical transfection methods such as lipofection or.
This was the first demonstration of efficient nonviral, liposomal delivery of both nucleic acids dna and sirna into hmscs. L final component per well 384well 96well 24well sirna 30 nm final 1. This reference provides general guidelines and procedures to transfect sirna. Reverse transfection is faster to perform than forward transfection and is the method of choice for high throughput transfection. 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. Advanced transfection with lipofectamine 2000 reagent. This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. The purpose of this study is to evaluate the effect of sirna transfection using lipo2000 on cellular functions and the possible underlying mechanism. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. We further provide evidence for successful sirna silencing of targeted gene rigi at both the mrna and protein levels. Effects of lipofectamine 2000sirna complexes on autophagy in. Another research group managed nonviral nucleic acid dna and sirna delivery in hmscs by using lipofectamine 2000 with a 50% efficiency hoelters et al. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
After incubation, i add the mixture 109 l to cells containing 0. Automated highthroughput sirna transfection in raw 264. Jun 10, 2011 lipofectamine 2000 is commonly used for sirna transfections. Thee two most important factors in choosing transfecting reagent are.
Lipofectamine 2000 transfection reagent from invitrogen. Older browsers that do not support html5 and the h. Lipofectamine 2000 from invitrogen biocompare product. Refer to the cell lines database for a list of cell types successfully transfected. L optimem medium for 5 min room temperature incubation. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Lipofectamine 3000 reagent for pluripotent stem cell applications. High efficiency, sitespecific transfection of adherent cells with sirna using microelectrode arrays mea the jove video player is compatible with html5 and adobe flash. To transfect cells with sirna, follow the protocol as described for dna but. Stealthsirna transfection protocol lipofectamine 2000. Lower cell densities are suitable with optimization of conditions.
Sep, 2005 both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. The main advantage of using lipofectamine 2000 however, was its ability to transfect a range of very different cell types coupled with a high efficiency of transfection. Sirna and plasmid cotransfection using lipofectamine 3000. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene. I used the protocol from the company and lipofectamin 2000. For each well to be transfected, prepare rnai duplex lipofectamine rnaimax complexes as follows. Lipofectamine 3000 protocol written out with timing. Dotap functionalizing singlewalled carbon nanotubes as. Lipofectamine 3000 reagent thermo fisher scientific. After 5 minutes incubation, combine the diluted dna with the diluted lipofectamine 2000 total volume is 500.
Lipofectamine 2000 transfection reagents highefficiency transfections. You can generally use other serumfree medium instead of optimem. Combine diluted lipofectamine 2000 cd reagent with diluted dna within 30 minutes. Enable high transfection efficiency in novel genome editing applications. We present our optimized high throughput nucleofection protocol as an efficient way of transfecting primary human monocytederived dendritic cells with either plasmid dna or sirna without causing cell maturation. Lipofectamine 3000 lipofectamine 2000 competitor fhd lipofectamine 2000 lipofectamine 3000 competitor 1 competitor 2 gststat. Plate cells so they will be 7090% confluent at the time of transfection.
Lipofectamine 2000 cd reagent thermo fisher scientific. Follow the web site instructions to further define the. L 5 5 incubate incubate for 5 minutes at room temperature. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Transfection protocol lipofectamine 3000 transfection reagent. Invitrogen lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Jove core jove science education jove lab manual high schools faculty resource center. In vitro differentiation of mature myofibers for live. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. If dmem is used as a diluent for the lipofectaminez 2000, mix with the diluted sirna within 5 minutes. For example, for cotransfection of plasmid dna and sirna in a 24well plate, we would recommend using.
I used 750000 cellswell in a 6well plate, got a 30% efficiency but most of the cells seemed unhealthy and almost dead 24 hours. Lipofectamine 2000 transfection reagent thermo fisher scientific. Effects of sirna interference of cdkl1 expression on. I tried reversetransfecting hepg2 cells using lipofectamine 2000. Effects of lipofectamine 2000sirna complexes on autophagy. I use lipofectamine 2000 as my standard transfection reagent of choice and found that it is just as efficient for simirna transfection compared to other reagents rnaimax, fugene, xtremegene. L cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Once several sirna sequences with high prediction scores are obtained basic. I wait 5 min and then combine the two tubes and wait 20 min. I am trying to cotransfect dna and sirna into hek cells. Singlestranded dna constructs can also be mixed with cas9 rnp complexes to promote homologydirected.
Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. Incubate the cells at 37c in a co 2 incubator for 2496 hours until you are ready to assay for gene knockdown. Do you have experience using an efficient transfection. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines.
Molecules free fulltext systematic screening of commonly. Jul 24, 2007 lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Optimized protocol for efficient transfection of dendritic. Lipofectamine 2000 reagent thermo fisher scientific. A number of factors affect this reagent, which must be considered before purchasing. After 5 minute incubation, combine the diluted dna. A common characteristic of cpps is the presence of highly basic amino acids, such as arginine and lysine, which suggests the importance of these cationic motifs in. Scaling up or down lipofectamine 3000 reagent transfections. If you own this web property, please visit our service catalog page to select a preference for handling your data otherwise you may continue to the site. The downregulation of gnao1 and its promoting role in. Transfection protocol lipofectamine 3000 transfection. In this study, we used three transfection reagents. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. Transfect plasmid dna and sirna at the same time using lipofectamine 2000.
Dilute sirna chimera with optimem i reduced serum medium invitrogen up to 50 l and vortex for a few seconds dilute appropriate amount of lipofectamine 2000 by optimem i reduced serum medium to 50 l and vortex for a few seconds for siha and skov3 cells, dilute 1. I tried to cotransfect plasmid dna and sirna silencer select from lifetechnologies into hek 293. In vivo sirna transfection and gene knockdown in spinal. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. Invitrogen lipofectamine 2000 transfection reagent life. 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 2000 is designed to be used with invitrogen media with the manufacturers recommending optimem for best dnareagent complex formation. This site is scheduled to expire on march 18, 2021 dartmouth. If you have difficulty in transfection efficiency you may also try reverse. To study the effect of sirna knockdown of the gene that is expressed by the cotransfected plasmid, transfection can be performed at same time by using one reagent, such as lipofectamine 3000 or lipofectamine 2000.
Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. How much time after sirna transfection by lipofectamine 2000. All amounts and volumes are given on a per well basis. Lipofectamine 2000sirna complexes cause endoplasmic. For other formats, see scaling up or down transfections. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. Transfection amounts component 96well 24well 6well final mrna per well 100 ng 500 ng 2500 ng final lipofectamine.
Lipofectamine rnaimax, what medium for transfection. Shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine 3000 reagent sirna lipofectamine 3000 reagent 96well 0. Lipofectamine 2000 is commonly used for sirna transfections. After the 5 minute incubation, combine the diluted sirna with the diluted lipofectaminez 2000 total volume is 100 l. Has anyone tried sirna transfection with lipofectamine3000. For some cell types, such as cos7, the transfection efficiency was over 95% while other hardertotransfect cells, such as caco2, displayed efficiencies around 75%.
Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Mix gently and incubate for 20 minutes at room temperature to allow the dna lipofectamine 2000 complexes to form. 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.
Jul 17, 2007 the manual also includes guidelines for scaling transfections up or down, e. High efficiency, sitespecific transfection of adherent cells. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Lipofectamine 2000 was gently mixed with sinc or sicdkl1 for 20 min room temperature incubation, and was then added into 6well plate. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine messengermax reagent protocol outline a. Dilute lipofectamine 3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Both, protein knockdown using sirnas as well as high and persistent. In addition, cell inhibition was assessed by a srb assay.
Stealthsirna transfection protocol lipofectamine 2000 thermo. After a 5 minute incubation in optimem, the lipofectamine 2000 and dna are combined and incubated for 20 minutes with gentle shaking at room temperature. How do you cotransfect dna and sirna into hek cells. To transfect cells with sirna, follow the protocol as. Although efficient delivery of dna and sirna can be achieved by cpps, the transfection efficacy rarely exceeds the commercially available standard of lipofectamine 2000 40. Lipofectamine stem reagent can be used to efficiently codeliver rnp complexes of cas9 protein with guide rna grna into ipscs, nscs, and mscs figure 7 to support high efficiency, targeted insertions or deletions indels. High transfection effi ciency enables lipofectamine 3000 to be used for a variety of applications, including reprogramming of fi broblasts, gene editing, and transfection of diffi cult cells. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Lipofectaminez 2000 with the diluted sirna within 30 minutes. Has anyone used lipofectamine 3000 for plasmid transfection. I used 40160 nm of dharmacon sirna and the ratio of lipofectamine. School of statistics, henan university of economics and law, zhengzhou 450046, china. In vivo sirna transfection and gene knockdown in spinal cord via rapid noninvasive lumbar intrathecal injections in mice the jove video player is compatible with html5 and adobe flash.
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