Today, several ways of introducing nucleic acid into cells are described. Transfection protocol on the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using invitrogen lipofectamine 3000 transfection reagent. Rnaimax reverse transfections lipofectamine thermo. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein.
Sigmas universal transfection reagent is a unique formulation of a proprietary polymer blend used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hardto. As discussed earlier, it is essential to invest time in optimizing all aspects of the screening pipeline. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rnai duplex and lipofectamine rnaimax concentrations. As nehad pointed out, you can do reverse transfection with lipofectamine 2000 or rnaimax. Plate cells so they will be 6080% confluent at the time of transfection. Prepare the transfection buffer as noted in the material section. Transfection reagent strong performance in a wide range of cell lines reliableone of the mostcited dna reagents strong performancefor easytotransfect to moderately difficulttotransfect cell lines easy to usesimple protocol for easy optimization in a wide range of cells invitrogen lipofectamine stem transfection reagent. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. Transfections allow for transient expression of a gene of interest in a target cell line and can be useful. Just follow the protocol for one experiment to get roughly an idea where to start then try to optimise the. How to perform sirna transfection with lipofectamine rnaimax protocol. Do not add antibiotics to media during transfection as this causes cell death.
Transfection protocol lipofectamine 3000 transfection. For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. The prepared mix is sufficient for triplicate transfections with overage. Optimizing knockdown conditions in the cell type of interest by transfecting a rnai. Reverse transfection and forward transfection protocols can be used for most cell lines tested. Unfortunately, no single delivery method or transfection reagent can be applied to all types of cells. Transfection types transient transfection and stable transfection duration. Goldman, prof atan gross lab, weizmann institute of science israel mef. Invitrogen lipofectamine rnaimax transfection reagent. Can anyone recommend a protocol for transfection of hepg2. Can anybody give me exact protocol for sirna transfection. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types.
Lipofectamine 2000, 3000, messengermax, and rnaimax transfection reagents green benefits sustainable packagingno dry or gel ice, no expanded polystyrene eps coolers introduction we are committed to designing our products with the environment in mindits part of how we enable our customers to make the world healthier, cleaner, and. Cells should be plated 18 to 24 hours prior to transfection so. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. The protocol indicated below is a reverse transfection protocol. The protocal is from thermo fisher scientific important guidelines for transfection 1. Reverse transfection an overview sciencedirect topics. Universal transfection reagent protocol sigmaaldrich.
Viafect transfection reagent has been shown to transfect cell lines. Cloned genes can be transfected into cells for biochemical characterization. Transfection of cas9 rnp ribonucleoprotein into adherent. Transfection is the introduction of dna, rna, or proteins into eukaryotic cells and is used in research to study and modulate gene expression. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Celltype specific transfection protocols are available at our website or through technical service. Mission sirna transfection reagent s1452 technical. The purpose of this protocol is to introduce dna or small rna into eukaryotic cells for recombinant expression or down regulation of defined target genes.
Can anybody give me exact protocol for sirna transfection using. Lipofectaminea rnaimax reagent thermo fisher scientific. Overnight transfection 6well plate format, standard protocol data from a. Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent. Lipofectamine rnaimax transfection reagent delivery. Lipofectamine 2000 and lipofectamine rnaimax transfection.
Chemical reagents one of the first chemical reagents used to transfer nucleic acids into cultured mammalian cells was deaedextran vaheri and. Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with excellent. Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. Gfp plasmid to sirna while doing lipofectamine 2000 transfection. There are several ways in which to introduce cas9guide rna complexes into cells. Plate cells to obtain 7080% cell density on the day of transfection. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Amounts of dna and reagent jetprime and competitors per well in 6well plate for transfection according to manufacturers recommendations. Transfection protocol on the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using invitrogen. This protocol is for a basic assay using an xcelligence rtca instrument to monitor the effects of sirna knockdown. Lipofectamine rnaimax reagents are cationiclipid transfection reagents formulated for superior transfection efficiencies on a variety of cell types. Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine transfection of c2c12 cells bridges lab. Transfection protocol lipofectamine 3000 transfection reagent.
The image below figure 1 is an example of the microarrays that can be created with this approach. Use this procedure to reverse transfect stealth rnai or sirna into mammalian cells in a 24well format for other formats, see. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine. Cells were transfected with lipofectamine rnaimax complexed with silencer select sirna at 30 nmwell. Lipofectamine rnaimax has a broad peak of activity. The prepared mix is enough to have triplicates 96well, duplicates 24well, and single well 6well transfections, and account for pipetting variations. Also prepare the sterile dna for transfection 2030 g10cm dish70 for a 15cm dish. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Timeline steps procedure details day 0 1 seed cells to be 60. For many adherent cell types, we have observed that lipofectamine rnai max from thermo gives close to optimal results and recommend it for starting. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration while reducing cytotoxicity in your experimental system. Reverse transfection protocol acea biosciences inc. Cookie information is stored in your browser and performs functions such as recognising you when you return.
Lipofectamine rnaimax offers a simple protocol no need to remove transfection complexes, or change or add medium following transfection. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. Lipofectamine rnaimax reagent thermo fisher scientific. As always, use good cell culture practices and wear your personal protective.
Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagentdna mix and assayed on day 2 or 3. How much lipofectamine rnaimax shoiuld be used for sirna. Rnaimax reverse transfections lipofectamine thermo fisher. 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. Cas9 nuclease may be used in vivo to create targeted genome modifications. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Thus, transfection techniques and protocols serve as an analytical tool that facilitates the characterization of genetic functions, protein synthesis, cell growth and development. Can anybody give me exact protocol for sirna transfection using rnaimax. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types.
Aug 06, 2009 transfection types transient transfection and stable transfection duration. Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Though this protocol has been optimized using the a549 human nonsmall cell lung cancer cell line, assay conditions may require addi. Lipofectamine 2000, 3000, messengermax, and rnaimax. Lipofectamine rnaimax transfection reagent delivery of. This protocol is optimized for transfection in a 6wellin.
Therefore, transfection rates and success between cell types may be more similar for sirnas than plasmid dna. The transfection is designed for one rna amount combined with one amount of lipofectamine rnaimax. Lipofectamine contains lipid subunits that can form. 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. We call this approach reverse transfection and we have developed two methods to reverse transfect cells. Transfection guide overview of transfection methods promega. Highest transfection efficiency in many cell types and formats e. In some cases, transfection must be optimized for different cell lines. A protocol for a novel transfection reagent designed to transfect dna into a wide variety of cell lines with high efficiency and low toxicity. The protocol type is indicated immediately under the title of the protocol.
Lipofectamine 2000, 3000, messengermax, and rnaimax transfection reagents green benefits sustainable packagingno dry or gel ice, no expanded polystyrene eps coolers introduction we are. Similarly, accuracy and reproducibility in preparing the plates for solidphase reverse transfection of. As always, use good cell culture practices and wear your personal protective equipment. See page 2 to view guidelines for transfection of sirnas using lipofectamine. Silencer select sirna transfection with lipofectamine rnaimax. To transfect cells with sirna, follow the protocol as described for dna but.
Use the following table to scale the volumes for your transfection experiment. 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. 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. 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. Mission sirna transfection reagent s1452 technical bulletin. Protocols based on wetlab experiments have provided successful results when used by qiagen scientists for transfection of the. Typical rnaimax transfection procedure transfect cells according to the following table.
We have found that lipofectamine rnaimax works well on the majority of cell lines that we have encountered and results in minimal cytotoxicity. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Scaling up or down lipofectamine 3000 reagent transfections. Transfection protocol transfection procedure this procedure is designed for sirna in combination with lipofectamine rnaimax. For additional information on scaling your transfection reaction, see page 4. Since this is a lot of rnaimax, it might be a good idea to first check it out in a. For plate size volume of sirna transfection reagent per well l reactions 1. Transfecting sirna using lipofectamine rnaimax pin. See page 2 to view a typical rnaimax transfection procedure. Because the cells are added directly to the dna, this process reduces the experimental time by one day and allows for highthroughput transfection of dna in a plate or microarray format.
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