Lipofectamine 2000 and lipofectamine rnaimax transfection. I want to know which cationic lipid and what polymers they have used. We recommend using invitrogen oligofectamine reagent when transfecting. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna.
Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Whats the difference between lipofectine and lipofectamine. The commonlyused plasmid dna or rna lipofectamine transfection reagent had been shown to be able to deliver many proteins into cells, so we tested several lipofectamine variants for their ability to efficiently deliver cas9 rnps sells et al. The intracellular trafficking mechanism of lipofectamine. 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. An example from the lipofectamine 2000 protocol is posted below. Liposome lipidbased formulation has the least influence on. 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 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. I have been doing research with primary rat cardiomyocytes for more than seven years, and plasmid transfection of these cells is not easy. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. How many screenings can be performed with the 1, 2. Dnalipofectamine 2000 cd complexes can be added directly to cells in culture medium. 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.
Despite this, a satisfactory mechanismbased explanation of their. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Comparison of transfection through lipofectamine and polyeth. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension.
Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Works effectively with all common cell lines as well as with many challenging ones, and can be used in media with or without serum. Canonical pathway enrichment analysis shows the primary biological processes impacted in hela cells that were transfected with transit. The the reagent provides high transfection efficiency in. Add the transfection mix dropwise being careful not to dislodge the cells. Comparison of transfection efficiency of nonviral gene transfer reagents. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine 2000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. Human crc cell lines hct116 and rko were transfected with mir140 mimic, inhibitor, or small interfering rna sirna against adamts5 or igfbp5, respectively, using oligofectamine or lipofectamine 2000. This is certainly true for transfections and different cell lines require the development of an efficient transfection protocol to optimize efficient delivery while. One 96well platetransfection reagent was used per cell line.
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. I use lipofectamine 2000 as my standard transfection reagent of choice and found that it is. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. If you are studying rnai oligos, the following link have different transfection protocol for different cell types. The sameday transfection and plating or reverse transfection will also help boost your percent transfected. Different sirna transfection agents have enyone tested. Transfection efficiencies lower than 50% were not reported in this table. Rapid and highly efficient mammalian cell engineering via. Optimizing transfection lipofectamine 2000 cd reagent. Versatile use of transfection reagents for short double. Mix gently and incubate for 20 minutes at room temperature solution may appear cloudy.
Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine 2000 transfection protocol 10cm plates reagents. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Cells were transfected with pegfp plasmid using either k2. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Complexes are stable for 6 hours at room temperature. Lipofectamine reagents go online to view related products. Formulation of lipofectamine 2000 and lipofectin signagen blog. Methods for sirnamediated reduction of mrna and protein.
Add 6l of lipofectamine2000 from biostores and 100 l of. The negative log of the p values is the probability of. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Expressfect, fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. With automated, highthroughput systems, we recommend a complexing volume of 50 l for transfections in 96well plates. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Invitrogen lipofectamine 2000 transfection reagent fisher. For the sirna or plasmid dna transfection of primary chondrocytes. 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. Plate cells so they will be 6080% confluent at the time of transfection.
Optimization procedure for small interfering rna transfection in a 384well format. Transittko, oligofectamine reagent, or lipofectamine 2000 diluted in serum free dmem. The following morning, carefully aspirate the media. Do you have experience using an efficient transfection reagent for. Not recommended for use with lipofectamine 2000 or lipofectamine rnaimax. In general, 3000 will have a better transfection efficiency 75% whereas 2000 may only be 4050%. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Different sirna transfection agents have enyone tested them.
Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Lipofectamine 2000, oligofectamine, fugene hd, and dharmafect2 were tested, each in accordance with the manufacturers instructions. Thee two most important factors in choosing transfecting reagent are. 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. Prewarm 50ml of optimum stored in cold room at 4c 10min. Increase transfection efficiency and cell viability with. Transittko, oligofectamine reagent, or lipofectamine 2000 diluted in serumfree dmem was applied to the dsrna mixture, and the formulation was continued for 15 min. Product is guaranteed for 6 months from the date of shipment if stored properly. Incubate the cells for 18 h, or until the following morning. Lipofection is most widely used method for transfecting neurons for livecell imaging experiments. Although invitrogen recommends their own brand of culture medium for transfection, called optimem, we found no difference between this and conventional media. We are using oligofectamine from invitrogen with great success. Downregulation enhanced green fluorescence protein gene.
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. Lipofectamine 2000 is a highperformance transfection reagent for gene expression and gene silencing. 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. An effective and efficient method of transfecting primary human. The amount of lipofectamine 2000 should be changed in proportion to the culture medium. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1. Incubate the cells 2448 h before checking for protein expression. Reagent is easy to use because it provides a simple and fast protocol. Invitrogen lipofectamine 2000 transfection reagent life. Invitrogen lipofectamine 2000 transfection reagent. Choosing the right transfection reagent for optimal efficiency. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Do you have experience using an efficient transfection.
Dnaplus lipid complexes must be made in serumfree medium such. Oligofectamine transfection reagent forms stable complexes with oligos, permitting. Oligofectamine transfection reagent thermo fisher scientific. Oligofectamine invitrogen was used for sirna transfection alone. Lipofectamine 2000 and rnaimax, both from invitrogen, gave the best results 90% efficiency in most of the cell lines i tested. Lipofectamine 2000 invitrogen was used in plasmid transfection and. Comparison of transfection efficiency of nonviral gene.
Lipofectamine 2000 reagent thermo fisher scientific. I previously believed that the best method was using lipofectamine 2000 with plus reagent and an overnight incubation with optimem. Lipofectamine 2000 transfection reagent dartmouth college. Most publications are using lipofectamine 2000 and oligofectamine invitrogen for rnai studies. Invitrogen transfection reagents are shipped on wet ice and should be stored at 4 c. It is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours. Ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems. 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. Immunohistochemistry was performed to determine the expressions of adamts5 and igfbp5 in crc tissues. Mirus lipofectamine 2000 and oligofectamine invitrogen. Mix each tube well by vortexing or flicking the tube. Transfection efficiency and cell toxicityviability, reagent dose response a dose response from.
Transfection guide overview of transfection methods. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Invitrogen lipofectamine 2000 cd transfection reagent 1ml. I use 24well plates for transfections in triplicates. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. For additional information on scaling your transfection reaction, see page 4. We examined the efficiency and cytotoxicity using nine different cell lines mc3t3e1 mouse preosteoblasts, pt30 human epithelial precancer cells. But efficiency does depend on the cell lines that you want to. Refer to the cell lines database for a list of cell types successfully. Pcr and realtime pcr, reverse transcription, cdna synthesis direct from cells. The results demonstrated that egfp expression was significantly and. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 is then added to culture medium within a sterile tube along with the dna at a 3.
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. Ideal for transient protein expression or highthroughput rnai transfections. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Invitrogen lipofectamine 2000 transfection reagent 15ml. Add the oligomerlipofectamine 2000 complexes to each well containing cells and medium. The vector contained an ofp reporter gene and was transfected. 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. Transfection reagent faqs thermo fisher scientific us. Lipofectamine 2000 from invitrogen biocompare product. Lipofectamine 2000 transfection reagent from invitrogen. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. This protocol may be useful in gene knockdown, and ectopic. Cell line successful transfection dharmafect formulation lipid a lipid b lipid c lipid d a549 v v bxpc3 v du 145. Incubate the cells at 37c in a co 2 incubator for 2496 hours until you are ready to assay for gene knockdown.
1320 722 772 833 1358 22 1107 1524 422 1153 29 121 445 827 727 1415 218 587 1098 1393 743 526 613 503 559 643 1327 1050 91 1475 217 629 1089 1226 1153 342 1237 290 345 138 1271