Transfection protocol lipofectamine 3000 transfection reagent. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Transfection protocol lipofectamine 3000 transfection. 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. For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. Transfection guide overview of transfection methods promega.
To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. It would be great if anyone can share a trusted protocol with me. Transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. No 20 is optimized for use with lipofectamine 2000, and is not recommended for lipofectamine rnaimax. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. 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. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. 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. 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. 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.
A sample protocol is listed here for transfection experiments performed in 6well plates. Transfection efficiency of p19 cells is between 3050%. Another advantage of lipofectamine 2000 is its application for cotransfection experiments in which plasmid dna and sirna are used simultaneously. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. From the luciferase activities and consistencies of.
Cells were transfected with a firefly luciferase encoding plasmid dna 0. Invitrogen lipofectamine 3000 transfection reagent fisher. Reverse transfection of stealth rnai or sirna using lipofectamine. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. 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. How does transfection with lipofectamine 2000 work. 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. Reverse transfection an overview sciencedirect topics. This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Rnaimax reverse transfections lipofectamine thermo. 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 of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used. Knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. Transfection protocol lipofectamine 3000 transfection reagent a549. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Transfection protocol lipofectamine 3000 transfection reagent mcf7. General guidelines for transfection follow these general guidelines when using lipofectamine 2000 to reverse transfect sirna or stealth rnai duplexes into mammalian cells. Reverse transfection on cell arrays for high content.
We have also used lipofectamine 2000 to introduce sirna into primary synoviocytes and obtained a. Transfection protocol lipofectamine 3000 transfection reagent mor. Luciferase activity was measured after 24 hours figures 24. Lipofectamine 2000 reagent thermo fisher scientific. Highest transfection efficiency in many cell types and formats e. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Transfection guide overview of transfection methods. Sep, 2005 transfection of primary synoviocytes resulted in greater than 50% transfection efficiency.
The use of dharmafect 2 and dharmafect 4 resulted in sirna uptake by a high proportion of bmdm, but caused considerable cell toxicity. Plasmid dna, synthetic sirna, rnai plasmids shrna, mir content and storage. Highthroughput applications, such as knockdown studies or target screenings, often include cell transfection. 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. 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. 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.
Comparison of small interfering rna sirna delivery into. 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. 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. 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. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
Blockit alexa fluor red fluorescent oligo manual for more. 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. 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. 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. Transfection efficiencies lower than 50% were not reported in this table. Max % of positive cells after transfection of a gfpplasmid 3. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. For each transfection sample, prepare oligomerlipofectamine 2000 complexes as follows. Cloned genes can be transfected into cells for biochemical characterization. Rnai in cultured mammalian cells using synthetic sirnas. Shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000. This protocol is for use with adherent cells, as transfection efficiency for.
To perform transfection experiments in other cell culture plates, simply multiply the. I tried reversetransfecting hepg2 cells using lipofectamine 2000. Transfecting sirna using lipofectamine rnaimax pin liu, 2017. Help on transfecting mcf7 and hct 116 cells transfection. Optimized delivery of sirna into 3d tumor spheroid cultures. 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. Page 2 gelatin preparation, dna purification and microarray printing page 2 cell preparation page 3 reverse. Ive been working with sirnas just for a couple of weeks and i have more doubts that answers. Reverse transfection of cells on plasmid or sirna arrays is a powerful alternative method to. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine. 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. 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.
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. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. 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. Cells are added directly to the lipofectamine 2000 rna complexes and transfection occurs while cells are attaching to the well. Has anyone used lipofectamine 3000 for plasmid transfection in. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Hiperfect, interferin, lipofectamine 2000, lipofectamine rnaimax, xtremegene and dharmafect 3 promoted good uptake of sirna by bmdm, without causing significant cell death. Rnaimax reverse transfections lipofectamine thermo fisher. 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. I use 24well plates for transfections in triplicates.
Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Reverse transfection is the transfer of genetic material into cells and is reverse because the. 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.
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. Reverse transfection instruction manual the two most powerful transfection technologies in one kit. Add 6l of lipofectamine2000 from biostores and 100 l of. In the protocol described here, we use a pool of three sirna duplexes per target gene per.
Reverse transfection of stealth rnai or sirna using. Reverse transfection page 2 and forward transfection page 3 protocols can be used. It is best to use polypropylene instead of polystyrene tubes. The protocal is from thermo fisher scientific important guidelines for transfection 1. Invitrogen lipofectamine 3000 transfection reagent. 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. Transfection was performed as described in protocol 2 with the following. I have used lipofectamine 3000 without p3000 to deliver sirna by reverse transfection, which is also. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Prewarm 50ml of optimum stored in cold room at 4c 10min. 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. Reverse transfection protocol for dna in 96well plates using transitx2 transfection reagent. Forward and reverse transfection protocols mirus bio.
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. 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 of nih3t3 cells, hela, swis 3t3, 293t with. Transfection reagents that can be used for reverse transfection. 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.