Transfection protocol lipofectamine 3000 transfection reagent mcf7. To perform transfection experiments in other cell culture plates, simply multiply the. Reverse transfection an overview sciencedirect topics. This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. Resuspend the sirna in h 2 o to a concentration of 100. Magnetofectamine is the association of the famous lipofectamine 2000 transfection reagent from invitrogen, with the unique magnetofection based reagent combimag, from oz biosciences list of magnetofectamine kits.
Version 1, 41 8 during the incubation of the printed slides with the transfection reagent, harvest hek293t cells and suspend 10 x 106 cells in 25 ml medium dmem with 10% ifs, 50 unitsml penicillin and 50 gml streptomycin. In the protocol described here, we use a pool of three sirna duplexes per target gene per. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. We have also used lipofectamine 2000 to introduce sirna into primary synoviocytes and obtained a comparable transfection efficiency when transfecting larger dna species scott et. Comparison of small interfering rna sirna delivery into. It is best to use polypropylene instead of polystyrene tubes. Introduction highthroughput parallel transfection techniques are a prerequisite to perform largescale experiments, such as genomewide rnai screens, in mammalian tissue culture cellss in such studies, transfections are typically performed in 96 or 384well plates using liquidhandling robotics. Transfection guide overview of transfection methods. Rnaimax reverse transfections lipofectamine thermo fisher. Cells were transfected with a firefly luciferase encoding plasmid dna 0.
Transfection guide overview of transfection methods promega. Reverse transfection of stealth rnai or sirna using lipofectamine 2000 in a 96. Transfection protocol lipofectamine 3000 transfection. Cloned genes can be transfected into cells for biochemical characterization. Reverse transfection protocol for dna in 96well plates using transitx2 transfection reagent. Blockit alexa fluor red fluorescent oligo manual for more. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. Highthroughput applications, such as knockdown studies or target screenings, often include cell transfection. Reverse transfection of plasmid dna automation is used for many applications to reduce variation caused by manual handling and to obtain reproducible results in highthroughput assays. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. I tried reversetransfecting hepg2 cells using lipofectamine 2000. Hiperfect, interferin, lipofectamine 2000, lipofectamine rnaimax, xtremegene and dharmafect 3 promoted good uptake of sirna by bmdm, without causing significant cell death. Transfection was performed as described in protocol 2 with the following.
Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells. Forward and reverse transfection protocols mirus bio. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Reverse transfection of cells on plasmid or sirna arrays is a powerful alternative method to. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al.
Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Genscript recommends using lipofectamine 2000 for all transfections. Transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. Our lab uses lipofectamine 3000 for transfections, but the only protocols for reverse transfection i can find on thermofisher scientifics website deal with their rnaimax reagent.
Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Has anyone here attempted reverse transfection using. Another advantage of lipofectamine 2000 is its application for cotransfection experiments in which plasmid dna and sirna are used simultaneously. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Optimized delivery of sirna into 3d tumor spheroid cultures.
It would be great if anyone can share a trusted protocol with me. Magnetofectamine is the association of the famous lipofectamine 2000 transfection reagent. Reverse transfection of stealth rnai or sirna using lipofectamine. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. May 21, 2018 c representative confocal images of ls174t spheroids showing localization of controlcy3 sirna formed in optimem using lipofectamine 2000 at 2, 6 and 24 hours post transfection.
However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used. 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. For each transfection sample, prepare oligomerlipofectamine 2000 complexes as follows. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0.
Invitrogen lipofectamine 3000 transfection reagent fisher. I use 24well plates for transfections in triplicates. Reverse transfection on cell arrays for high content. Rnai in cultured mammalian cells using synthetic sirnas. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. The protocal is from thermo fisher scientific important guidelines for transfection 1. How does transfection with lipofectamine 2000 work. Apr 15, 2014 the use of dharmafect 2 and dharmafect 4 resulted in sirna uptake by a high proportion of bmdm, but caused considerable cell toxicity. To test the transfection efficiency of the reverse transfection approach, a luciferaseencoding plasmid was reverse transfected into hek293, hep g2 and jurkat cells using fugene 6, fugene hd, transitlt1 and lipofectamine 2000 transfection reagents. Reverse transfection instruction manual the two most powerful transfection technologies in one kit. This protocol is for use with adherent cells, as transfection efficiency for.
Max % of positive cells after transfection of a gfpplasmid 3. Reverse transfection of stealth rnai or sirna using. Knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. From the luciferase activities and consistencies of.
Transfection protocol lipofectamine 3000 transfection reagent mor. 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. Luciferase activity was measured after 24 hours figures 24. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine. For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. Transfection protocol of lipofectamine rnaimax can be. C representative confocal images of ls174t spheroids showing localization of controlcy3 sirna formed in optimem using lipofectamine 2000 at 2, 6 and 24 hours post transfection.
If the nucleic acid to be transfected is immobilized or spotted in a multiwell format already as in the case of cdnashrnasirna screens, the transfection protocol is referred to as solid phase reverse transfection. Transfection efficiencies lower than 50% were not reported in this table. Reverse transfection of cells on plasmid or sirna arrays 5,6,7,8,9,10 is a powerful alternative method to perform highthroughput transfections for phenotypic data acquisition by light. Has anyone used lipofectamine 3000 for plasmid transfection in.
Cells are added directly to the lipofectamine 2000 rna complexes and transfection occurs while cells are attaching to the well. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. I have used lipofectamine 3000 without p3000 to deliver sirna by reverse transfection, which is also. Page 2 gelatin preparation, dna purification and microarray printing page 2 cell preparation page 3 reverse transfection of printed microarrays page 4 detection methods page 5 immunofluorescence page 6 autoradiography page 6 western blots page 6 tips and information page 7 alternative method. Co transfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0.
Transfection protocol lipofectamine 3000 transfection reagent. If the nucleic acid to be transfected is immobilized or spotted in a multiwell format already as in the case of cdnashrnasirna screens, the transfection protocol. Transfecting sirna using lipofectamine rnaimax pin liu, 2017. Lipofectamine 2000 reagent thermo fisher scientific.
Introduction highthroughput parallel transfection techniques are a prerequisite to perform largescale experiments, such as genomewide rnai screens, in mammalian tissue culture cellss in such studies. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Ive been working with sirnas just for a couple of weeks and i have more doubts that answers. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. 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. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection efficiency of p19 cells is between 3050%. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. 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. The use of dharmafect 2 and dharmafect 4 resulted in sirna uptake by a high proportion of bmdm, but caused considerable cell toxicity. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very. Rnaimax reverse transfections lipofectamine thermo. We have also used lipofectamine 2000 to introduce sirna into primary synoviocytes and obtained a.
Reverse transfection of cells on plasmid or sirna arrays 5,6,7,8,9,10 is a powerful alternative method to perform highthroughput transfections for phenotypic data acquisition by light microscopy. Plasmid dna, synthetic sirna, rnai plasmids shrna, mir content and storage. A sample protocol is listed here for transfection experiments performed in 6well plates. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. Sep, 2005 transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. Highest transfection efficiency in many cell types and formats e. From the luciferase activities and consistencies of data using different transfection reagents including genejammer, effectine, wellfect, lipofectamine, what i can say from the data i observed, the efficiency of transfection is much more better in hct116 cells. Version 1, 41 8 during the incubation of the printed slides with the transfection reagent, harvest hek293t cells and suspend 10 x 106 cells in 25 ml.
To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Page 2 gelatin preparation, dna purification and microarray printing page 2 cell preparation page 3 reverse. Reverse transfection page 2 and forward transfection page 3 protocols can be used. Invitrogen lipofectamine 3000 transfection reagent. Advanced transfection with lipofectamine 2000 reagent. Transfection reagents that can be used for reverse transfection. Transfection protocol lipofectamine 3000 transfection reagent a549. According to the manufacturer, this step is not necessary due to low cytotoxicity of the reagent, which surely makes the use of lipofectamine 2000 even more convenient. 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.
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. Add 6l of lipofectamine2000 from biostores and 100 l of. Mar 27, 2007 according to the manufacturer, this step is not necessary due to low cytotoxicity of the reagent, which surely makes the use of lipofectamine 2000 even more convenient. General guidelines for transfection follow these general guidelines when using lipofectamine 2000 to reverse transfect sirna or stealth rnai duplexes into mammalian cells. Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. Lipofectamine 2000 transfection reagent from invitrogen. No 20 is optimized for use with lipofectamine 2000, and is not recommended for lipofectamine rnaimax.