Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Reverse transfection is faster to perform than forward transfection and is the method of. After removal of the transfection reagent, the slide is placed in a culture dish and covered with cells in media. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. After 4h aspirate media and refeed cells with normal media. Lipofectamine transfection of c2c12 cells bridges lab. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. In standard transfection, sirna and lipid are complexed and added to preplated cells figure 1a. Lipofectamine rnaimax reagent protocol thermo fisher scientific. Invitrogen lipofectamine rnaimax is a proprietary formulation specifically. A sample protocol is listed here for transfection experiments performed in 6well plates. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start.
Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Cells in a 6 well dish, plated and at 70% confluence. For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. To investigate the influence of medium and transfection time, primary hepatocytes were transfected with metafecte pro, lipofectamine 2000 or targefect in the presence of dmem figure 2a or optimem, withwithout virofect enhancer, for 3 hours or 16 hours figure 2a, 2b, 2c. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. Knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. Ive been working with sirnas just for a couple of weeks and i have more doubts that answers. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Help on transfecting mcf7 and hct 116 cells transfection. Transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. Highthroughput applications, such as knockdown studies or target screenings, often include cell transfection. 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. Lipofectamine 2000 invitrogen, catalog number 11668019.
Add 6l of lipofectamine2000 from biostores and 100 l of. Transfection was performed as described in protocol 2 with the following modifications. So i asked one of my lab mates and he told me to use just 1ul. Reverse transfection and forward transfection protocols can be used for most cell lines. Transfecting sirna using lipofectamine rnaimax pin liu, 2017. In the protocol described here, we use a pool of three sirna duplexes per target gene per. Lipofectamine rnaimax reagent offers an advanced, efficient solution for sirna or mirna delivery. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Reverse transfection of stealth rnai or sirna using. Transfection protocol lipofectamine 3000 transfection reagent mor. Reverse transfection an overview sciencedirect topics.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. They differ in the order and timing of the addition of the three necessary components of transfection. Forward and reverse transfection protocols mirus bio. 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. Lipofectamine 3000 reagent outperforms lipofectamine 2000 and. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine. Transfection protocol lipofectamine 3000 transfection reagent gct. Has anyone here attempted reverse transfection using.
Work flow for multiplexing sirna assays by solidphase reverse. Transfection reagents that can be used for reverse transfection. 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. 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. Schematic overview of the reverse sirna transfection protocol.
Other reagents did not demonstrate effective transfection to this cell line. How does transfection with lipofectamine 2000 work. Lipofectamine 2000 worked better than three other transfection reagents we. Reverse transfection on cell arrays for high content. Reverse transfection instruction manual the two most powerful transfection technologies in one kit. Reverse transfection of 2 x 105 cells per well of a 24well plate with. An sirnabased method for efficient silencing of gene expression in. The presence of optimem resulted in significantly higher transfection rates when lipofectamine 2000 was used. 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. Luciferase activity was measured after 24 hours figures 24. C representative confocal images of ls174t spheroids showing localization of controlcy3 sirna formed in optimem using lipofectamine 2000. Combine the two volumes of optimemdna and optimemlipofectamine. Sep, 2005 transfection of primary synoviocytes resulted in greater than 50% transfection efficiency.
When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. The transfected cell microarray forms in 12 days and is then ready for downstream assays. Transfection protocols for forward and reverse transfection applications. 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. No other transfection reagent for rna interference rnai. 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. It is best to use polypropylene instead of polystyrene tubes.
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. Another advantage of lipofectamine 2000 is its application for cotransfection experiments in which plasmid dna and sirna are used simultaneously. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Prewarm 50ml of optimum stored in cold room at 4c 10min. 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. Comparative nucleic acid transfection efficacy in primary. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. So i asked one of my lab mates and he told me to use just 1ul lipofectamine for 20pmol of sirna thats the protocol specificationsbut instead for each well, use that amount for three wells. Lipofectamine 2000 transfection reagent from invitrogen. 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. Optimized conditions for successful transfection of human. Pdf solidphase reverse transfection on cell microarrays is a highthroughput method for the.
No 20 is optimized for use with lipofectamine 2000, and is not recommended for lipofectamine rnaimax. The protocal is from thermo fisher scientific important guidelines for transfection 1. Higher densities can lead to spontaneous differentiation. 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 order of dna and cells is reverse that of conventional transfection. Transfection protocol lipofectamine 3000 transfection reagent a549. On the day of transfection, complexes are prepared in a multiwell plate and incubated for the recommended complex formation time. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. Lipofectamine plasmid transfection bridges lab protocols.
Invitrogen lipofectamine 3000 transfection reagent. Shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine 3000 reagent sirna lipofectamine 3000 reagent 96well 0. Detailed reverse transfection protocols for plasmid dna and sirna can be. Genscript recommends using lipofectamine 2000 for all transfections. I use 24well plates for transfections in triplicates. Invitrogen lipofectamine 3000 transfection reagent fisher. Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. 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. Reverse transfection and forward transfection protocols can be used for most cell lines tested. 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.
Lipofectamine 2000 transfection reagent dartmouth college. For the amounts of transfection reagent i follow the manufacturers instructions and my sirna. Plasmid dna, synthetic sirna, rnai plasmids shrna, mir content and storage. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used.
Rnaimax reverse transfections lipofectamine thermo. Maintain p19 cells undifferentiated in mem with 10% serum 7. 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. Transfection and genome engineering fisher scientific. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Pdf work flow for multiplexing sirna assays by solidphase. Reverse transfection combines rna, transfection reagent, and cells in an altered sequence compared to traditional lipofectamine 2000 transfection protocols. 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. Transfection protocol lipofectamine 3000 transfection reagent. Lipofectamine rnaimax reagent thermo fisher scientific. Transfection protocol lipofectamine 3000 transfection reagent ncih522. The method illustrated is the gelatindna method of the reverse transfection approach. Max % of positive cells after transfection of a gfpplasmid 3.
Highest transfection efficiency in many cell types and formats e. Rnaimax reverse transfections lipofectamine thermo fisher. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. 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. Freshly passaged cells are then directly added to the. 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. Optimized delivery of sirna into 3d tumor spheroid cultures. Transfection protocol lipofectamine 3000 transfection. Cells were transfected with a firefly luciferase encoding plasmid dna 0. 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. For many adherent cell types, we have observed that lipofectamine rnai max from. 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.
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