Can i use the lipefectamine2000 for mimic mirna transfection. Mir8 downregulates mirna processing in hela cells by. Lipefectamine2000 can transfer dna and sirna, so it may be not perform as well as in. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. View our transfection products at fisher scientific. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Media were replaced with antibioticsfree media 612 h before 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. I use 24well plates for transfections in triplicates. In order to study mirna function in human adipocytes, we aimed for the modulation of mature mirna concentration in these cells. Comparative nucleic acid transfection efficacy in primary. Deliver microrna mirna effectively with transitx2 transfection reagent. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.
Lentiviral mirna expression plv mirna expression vector system. Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with excellent. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. Refer to the instructions provided with your transfection agent for the recommended volume. Thee two most important factors in choosing transfecting reagent are. 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.
Genome editing, stem cell manipulation, and immunotherapy are a few of the many. Lipofectamine rnaimax reagent protocol thermo fisher scientific. 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. Despite having a specific protocol, the volume of this reagent should be optimized for use in. Transfection of microrna mimics should be used with caution.
Co transfection protocol for plasmid dna and sirna mirna. Both lipofectamine 2000 and lipofectamine rnaimax reagents are cationiclipid transfection reagents formulated for superior transfection efficiencies on a variety of cell types. The comparison between primary and metastatic cancer tissues was also accessed. Lipofectamine 2000 is a transfection reagent with liposome structure. Generally, transfection reagents that can deliver smaller. Invitrogen lipofectamine rnaimax transfection reagent. Abstract microrna induced gene regulation is a growing field in basic and translational research. Concentration of dna for stable transfection with lipofectamine 2000 and the per transfection and transduction. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Stealthsirna transfection protocol lipofectamine 2000. Cotransfection protocol for plasmid dna and sirnamirna. Transfection amounts per well use 10 nm mirna duplex as a starting point.
On measuring mirnas after transient transfection of mimics. Advanced transfection with lipofectamine 2000 reagent. The other thing is that ive seen that in lipofectamine 2000 manual there are two different protocols depending if you are using a vector or a sirna. Microrna107 is a novel tumor suppressor targeting pou3f2. Comparative transfection efficiency of jetprime versus its main competitor. Lipofectamine 2000 transfection reagent from invitrogen. One plasmid contains mirna precursor and the other, 3utr target.
Each reporter plasmid ng cells well total volume well. Lipofectamine 2000 is primarily used for delivery of plasmid dna, but it also works well for delivery of sirna and mirna mimics. Do you have experience using an efficient transfection. Refer to the cell lines database for a list of cell types successfully transfected. Applications sirnamediated gene silencing protein regulation with mirna highthroughput screening. The data indicated that lipofection protocol could transfect mirnas into. Transfection efficiencies were assessed by luciferase activity rlu. For mirna transfection, follow the user protocol and simply substitute duplex. Lipofectamine 2000 and lipofectamine rnaimax transfection. Invitrogen lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. The amounts indicated result in a final oligonucleotide concentration of 30 nm. See page 2 to view a typical rnaimax transfection procedure. Dna complexes were added to cells that had already been allowed to attach to the plate b. Cotransfection of two plasmids tissue and cell culture.
Lentivectorbased microrna precursor constructs cat. Can i use the lipefectamine 2000 for mimic mirna transfection. Sbis mirna construct can be used for transient expression of mirna using conventional transfection protocols. I used to use lipofectamine 2000 from invitrogen, but recently i use micoporatormp100 for both mirna and plasmid dna transfection, which gives me better results 90% efficiency in the most. Lipofectamine 2000 transfection reagent were used to transfect premir. Shop online for a wide selection of invitrogen lipofectamine rnaimax transfection reagent provides highest transfection efficiencies on widest variety of cell types for. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Cells were first plated into 24well plates followed by lipofectamine 2000 mediated transfection with these top flash or fop flash constructs. Htm cells were transfected using lipofectamine with a fluorescent mirna dy547 and analyzed 48 hours after transfection. Universal polymer reagent for delivering dna, sirna, mirna and crisprrnp to many cell lines. Co transfection of two plasmids need the protocol for lipofectamine 2000 based transfection nov 012009 hi, i am trying to cotransfect transient trasnfection two different plasmids in to hek293t cells.
Is this amount ok for mirna and sirna transfections. Highest transfection efficiency in many cell types and formats e. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. To transfect cells in different tissue culture formats, vary. Co transfection of plasmid dna and sirna mirna is a technique popularly used by cell biologists, particularly to assess sirna mirna mediated knockdown of gene expression from a plasmid dna that is being delivered to the cell. Synthetic mirna mimics from dharmacon or shanghai genepharma were transfected into cells following the dharmafect1 transfection protocol t200103. Instead the vast majority of mirna transfected with either hiperfect, fig. This protocol is optimized for transfection in a 6well culture plate format to perform transfection in different cell. Microrna520f3p inhibits proliferation of gastric cancer. Deliver microrna mirna effectively transfection, electroporation.
The average lentiviral titers in our preparations are around 56x 10 5 x 10. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Once reconstituted in nucleasefree water, the mirna is ready to transfect and can be used at your choice of final concentration. Establishment of lipofection protocol for efficient mir21. This protocol is to be used to test the binding and activity of mirna on putative utr target sequences. In this video, we will perform an sirna transfection experiment using. Quantitative pcr following mirna mimic transfection or addition post lysis. Lipofectamine 2000 transfection reagent dartmouth college. Establishment of lipofection for studying mirna function.
Moreover, its lentiviral backbone allows each mirna construct to be packaged in pseudoviral particles and introduced into. Read advanced transfection with lipofectamine 2000 reagent. Rnai transfection protocol see page 2 to view guidelines for transfecting mirnas using lipofectamine rnaimax reagent. Lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The presence of optimem resulted in significantly higher transfection rates when lipofectamine 2000 was used. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. First, we evaluated the expression of mir107 in melanoma cells and tumor tissues. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. Endofectin transfection reagent, cell lines genecopoeia. For each well to be transfected, prepare mirna lipofectaminetm rnaimax complexes as follows. A comparison showing transfection efficiency of polyjet reagent vs. Lipofectamine 2000 right panel around 5% efficiency.
We hereby aimed to characterize a novel microrna mir in the metastatic melanoma model. Transitx2 can also be used for cotransfecting plasmid dna and mirna. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Cloned genes can be transfected into cells for biochemical characterization, mutational. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. B vector according to the manufacturers protocol invitrogen and then. Does anyone have a protocol for microrna transfection of. Cell lines were transfected with a firefly luciferaseexpressing plasmid. On measuring mirnas after transient transfection of mimics or antisense inhibitors.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. The mirna of interest can be delivered into cells by transient transfection of the plv mirna. Ca were cotransfected into hek293t cells as previously described 3. The first one is that for transfections in hela cells when i was using promoter vectors, i used to add around 70. Therefore, the high level of transfected mirna detected by qpcr is largely attributable to their. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. Examining this regulation directly in cells is necessary to validate high. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations.
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