Mar 14, 2020 lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the or contact technical services for other specialized transfection protocols. Transfection protocol sirna resuspension guidelines we recommend preparing 10. We recommend to use sirna chimera at a concentration lower than 5 nm, and where possible, at 1 nm. I use 24well plates for transfections in triplicates. In separate tubes, dilute the sirna tube 1 and the appropriate dharmafect transfection reagent tube 2 with serum free medium. The protocal is from thermo fisher scientific important guidelines for transfection 1. In a six well tissue culture plate, seed 2 x 105 cells per well in 2 ml anti bioticfree normal growth medium. There is no much difference between transfecting sirna and plasmid. Transfer 10 l of the fs in an rnase free micro reaction tube and sonicate it in.
Genscript recommends using lipofectamine 2000 for all transfections. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Rnai transfection protocols thermo fisher scientific uk. Indeed, some mirna are also known to induce gene expression by association with the promoter of the gene of interest. The unique properties of the delicate cells make transfection challenging and necessitate specialized techniques. Once the sirna is configured against the gene it has to be effectively delivered through a transfection protocol. Dna, rna, or protein using one of various transfection methods, like lipofection, electroporation, calcium phosphate transfection, liposome transfection, and viral transduction, as well as physical methods such as microinjection and biolistic particle delivery. We are currently having technical issues with our pdf. Mammalian cell penetration, sirna transfection, and dna. Briefly centrifuge the plate to ensure that the dried sirna is at the bottom of the wells. Application protocol using transfection for sirna delivery.
Or you can change serum containing medium to a serum free medium after 624 hrs of sirna transfection. Resuspend the sirna in nuclease free water at a final concentration of 10. Several sirna delivery systems and reagents are available depending on the cell. In this protocol reverse transfection of hepg2 cell is recommended. We provide many free cell linespecific transfection protocols in the cell line database. Briefly centrifuge tubes or multiwell plates containing sirna to ensure that the sirna pellet is located at the bottom of the tube. This protocol may also serve as guidelines for microrna and plasmid co transfection. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Celltype specific transfection protocols for stealth rnai and sirna. Mcf7 cells were transiently transfected in 24well plates with different volumes of silentfect lipid reagent, 0.
Add 1 ul lipofectamine 2000 into 30 ul dmem without serum, mixing well and wait for 5 min at room temperature. A standard protocol for sirna transfection of mammalian cells rockville md 20850 100 l 500 l l 9601 medical center drive fax. Transfection protocols are used in biological laboratories for introduction of foreign plasmid dna, small therapeutic rna, or protein molecules into cells of a eukaryotic nature. The sirna transfection reagent is vided as a sterile solution, and is compatible with serum and antibiotics.
Rnai transfection intereference rnai sirna transfection. Brochure detailing reagents for efficient and robust dna and rna transfection. Transfection the transfer of genetic material into mammalian cells is a valuable tool that allows scientists to genetically manipulate neurons and neuronal tissue. For transfection procedure, follow protocol provided by transfection reagent manufacturer.
Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Mission sirna transfection reagent s1452 technical bulletin. We recommend to use sirnachimera at a concentration lower than 5 nm, and where possible, at 1 nm. In this technique sirna first must be designed against the target gene. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. You can transfect the cells in penstrep free medium or serum free medium depending the transfection reagent you use. How to perform sirna transfection with lipofectamine rnaimax protocol. Promega corporation 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mrna after transcription, preventing.
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Transit tko transfection reagent protocol for mir 2150, 2154, 2155, 2156 page 3 of 11 additional tips for dna and sirna cotransfection observe the following recommendations in addition to the tips for sirna transfection when performing a co transfection of dna and sirna. Pepmute sirna transfection reagent is a novel peptide based sirna. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Resuspend the sirna in nucleasefree water at a final concentration of 10. L volume of diluted dharmafect transfection reagent in serumfree medium.
Use this brief procedure to transfect stealth rnai or sirna into mammalian cells in a 24well format. L volume of the sirna in serum free medium by adding 0. Greater understanding of how rnai works is an excellent research tool that experts have utilized in the development of pharmaceuticals as well as therapies in medicine, offering researchers the ability to literally stop specific gene activity in its track. Dna in the optimem or a serumfree medium to bring the volume to 220. Repeated auroraa sirna transfection results in effective apoptosis of a549 cells compared to single transfection. Stealthsirna transfection protocol lipofectamine 2000. Small interfering rna sirna, sometimes known as short interfering rna or silencing rna, is a class of doublestranded rna noncoding rna molecules, 2025 base pairs in length, similar to mirna, and operating within the rna interference rnai pathway. Dissolve sirnas to a convenient stock concentration using the recommended volume of depcdw or rnase free water shown in table 1. Lipofectamine 2000 reagent thermo fisher scientific.
L transfection of sirna to transfect cells with sirna, follow the protocol as described for dna but do not add p3000 reagent when diluting the sirna step 3. Relaxing tea jazz elegant intrumental jazz music for work,study,reading relax music 4,195 watching live now. A sample protocol is listed here for transfection experiments performed in 6well plates. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. The protocol does not require removal of serum or culture medium and does not. Transfection amounts per well use 10 nm sirna duplex as a starting point.
This protocol describes a method for efficiently transfecting sirna in freshly isolated human villous cytotrophoblasts using microporation and identifying dnaprotein complexes in these cells. Stealth rnai and sirna transfection concentrations the transfection concentration of a stealth rnai or sirna duplex is determined by dividing the number of moles of sirna used by the final volume of the transfection i. I suggest that you follow the protocol for hepg2 sirna transfection introduced by life technologies company. At 48 hours posttransfection the cells were analyzed by qrtpcr for lamin ac gene expression levels. For optimal transfection conditions with the mission sirna transfection reagent, the for 1. On the day of transfection just prior to preparing complexes, plate 1. Transfection is the introduction of any nucleic acid molecule by nonviral means into cultured eukaryotic cells.
Dharmafect duo transfection reagentfor sirna and plasmid. Silencing of gene expression in cultured cells using small. Silencer select predesigned, validated, and custom sirna. Overview of adherent cell transfection protocol for a 96well plate. In order to avoid interfering endogenous mirna network, it is important to keep the sirna chimera transfection concentrations as low as possible. Transfecting sirna using lipofectamine rnaimax pin. Depending on the cell line and cell density the dharmafect.
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. The risc sirna complexes catalyzes cleavages and degradation of complement mrna molecules. In the past, it was often used only in reference to dna, but that has changed as applications such as rnai and more recently, crispr have been developed. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng final lipofectamine 2000 reagent per well 0. Rnai transfection protocols thermo fisher scientific us. While sirna provides the opportunity to induce gene knockdown in a variety of cell lines, their utility is limited to cells that are amenable to transfection of synthetic oligonucleotides. Lipofectamine rnaimax reagent protocol thermo fisher scientific. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. It may also refer to other methods and cell types, although other terms are often preferred. Rnai transfection protocol see page 2 to view guidelines for transfecting sirnas using lipofectamine rnaimax reagent. For cotransfection with both rna and plasmid dna, please follow the protocol in section iii. In animal cells, transfection is the preferred term as. One day prior to transfection, plate cells in 1 ml of complete growth medium so that.
Add 4 l of transpass hela transfection reagent to 100 l of serum free medium e. Table 4 provides a range of dharmafect volumes for alternate plate. Dharmafect transfection reagentssirna transfection protocol. Please feel free to contact cst with any questions. Protocol dharmafect duo transfection reagentfor sirna and plasmid co transfection this protocol is optimized for use with 100 ngwell plasmid and 100 nm sirna in a 96well plate format. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.
Table 1 presents recommended conditions for the most efficient delivery and subsequent silencing of gapdh and cyclophilin b sirna in various cell lines. Promega corporation 2800 woods hollow road madison, wi 537115399 usa toll free in usa 8003569526 6082744330 fax 6082772516 5. Mission sirna transfection reagent s1452 technical. Once reconstituted in nuclease free water, the sirna is ready to transfect and can be used at your choice of final concentration.
Prepare cells for transfection 12well plate format. Lipojet in vitro dna and sirna transfection kit a general protocol for transfecting mammalian cell 100 l toll free. For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. Can anyone help with hepg2 cells sirna transfection. Rna interference rna interference rnai rna interference is a process by which the expression of double stranded rna specifically stimulate a cell process that reduce gene expressions in a sequence specific manner. Delivery is usually done by cationic liposomes, polymer nanoparticles, and lipid conjugation. Introduction delivery of exogenous small interfering rna sirna in cultured cells is an effective method to modulate target gene expression via rna interference rnai. 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. Transfection of cells with rna use this protocol to transfect cells with rna only. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Nov 18, 2016 relaxing tea jazz elegant intrumental jazz music for work,study,reading relax music 4,195 watching live now. Common approaches include transfection of different substrates e. The following is a general protocol for use of dharmafect transfection reagents to deliver sirna into mammalian cells and was optimized for adherent cells in a 96well plate format. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products.
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