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