당신은 주제를 찾고 있습니까 “dharmafect duo protocol – siRNA Transfection Protocol“? 다음 카테고리의 웹사이트 Chewathai27.com/you 에서 귀하의 모든 질문에 답변해 드립니다: Chewathai27.com/you/blog. 바로 아래에서 답을 찾을 수 있습니다. 작성자 Thermo Fisher Scientific 이(가) 작성한 기사에는 조회수 85,273회 및 좋아요 334개 개의 좋아요가 있습니다.
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dharmafect duo protocol 주제에 대한 동영상 보기
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d여기에서 siRNA Transfection Protocol – dharmafect duo protocol 주제에 대한 세부정보를 참조하세요
Learn more at http://www.lifetechnologies.com/transfection
How to perform siRNA transfection with Lipofectamine® RNAiMAX protocol. Superior siRNA/miRNA delivery and gene knockdown.
In this video, we will perform an siRNA transfection experiment using Lipofectamine® RNAiMAX reagent.
As always, use good cell culture practices and wear your personal protective equipment. Clean the cell culture hood and work surface by spraying and wiping them down with 70% ethanol.
The day prior to your transfection, seed your cells so that they will be 60% to 80% confluent at the time of your experiment.
The day prior to your transfection, seed your cells so that they will be 60% to 80% confluent at the time of your experiment.
For this transfection experiment you will need:
– Lipofectamine® RNAiMAX reagent
– Opti-MEM® Reduced-serum Medium
– siRNAs diluted to a working concentration of 10 micromolar. We will be using two Ambion® Silencer® Select siRNAs, a BLOCK-iT™ Alexa Fluor® red fluorescent siRNA, and a negative control siRNA
– Five, 1.5 milliliter microcentrifuge tubes in a rack
– A P200 and P10 pipette and appropriate tips
– A marker and a timer
– And a 24-well plate with 60% to 80% confluent cells
We will be following the 24-well plate format of the Lipofectamine® RNAiMAX protocol.
Because we have 4 siRNAs, we will prepare a master mix of RNAiMAX. Add 200 microliters of Opti-MEM® medium and 12 micoliters of RNAiMAX in a tube labelled \”Master Mix\”
Mix well by vortexing or flicking the tube.
Add 50 microliters of Opti-MEM® Medium into each of 4 tubes and label them 1, 2, Positive, and Negative.
Add 3 microliters of each 10 micromolar siRNA stock to its corresponding tube. Mix well.
Add 50 microliters of the RNAiMAX master mix to each of the siRNA dilutions in tubes 1, 2, Positive, and Negative.
Incubate the complexes for 5 minutes at room temperature.
After the 5-minute incubation, remove your 24-well plate containing your cells from the incubator and bring it to the workspace in the hood.
Add 50 microliters of the siRNA-reagent complex from tubes 1, 2, Positive, and Negative to wells 1 to 4 of the 24-well plate, respectively.
You should have enough volume to run duplicates if desired.
Place your 24-well plate back into the incubator and grow cells for 1 to 3 days at 37 Celsius.
After incubating the cells 24 hours at 37 degrees Celsius, assess the transfection efficiency of the BLOCK-iT™ Alexa Fluor® red fluorescent siRNA using the FLoid cell imaging station or microscope.
To assess gene knockdown use a quantitative method such as Ambion® Cells-to-Ct™ Kit and Real-Time PCR.
For transfection protocols, FAQs, troubleshooting, and tips \u0026 tricks visit http://www.lifetechnologies.com/transfection
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Dharmacon™ DharmaFECT™ 1–4 transfection protocol – Cultek
The following is a protocol for transfecting Dharmacon™ synthetic siRNA or microRNA reagents into cultured mammalian cells using DharmaFECT™.
Source: www.cultek.com
Date Published: 12/27/2021
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Thermo Scientific Dharmacon® DharmaFECT® Duo …
DharmaFECT® Duo is a lip-based reagent specially formulated for co-transfection of plasm and siRNA. Under optimized conditions, efficient delivery of both.
Source: switchgeargenomics.com
Date Published: 5/20/2022
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Protocol
This protocol uses DharmaFect Duo transfection reagent (Dharmacon). DAY 1. Goal: Seed cells to yield ≥80% confluence at time of transfection.
Source: www.biocat.com
Date Published: 6/16/2022
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Thermo Scientific DharmaFECT Transfection Reagents
DharmaFECT. Duo protocol available. Human. A549. Lung carcinoma. 1. 0.2. 1 x 104. 2, 3, 4. X. BxPC3. Pancreas adenocarcinoma. 2. 0.2. 5 x 103. 1, 3, 4. DU …
Source: www.fishersci.com
Date Published: 1/10/2021
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DharmaFECT™ Transfection Reagents – encode
The following is a general protocol for use of Dharmacon DharmaFECT transfection … DU 145. Prostate carcinoma. 1. 94. 0.2. 1 × 104. 2, 3, 4. NCI-H1299.
Source: www.encodeproject.org
Date Published: 6/29/2022
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Enrichment of transfected cells with Dharmacon Edit-R …
DharmaFECT™ Duo transfection reagent (1 mg/mL; Cat #T-2010-xx) … The following is a general protocol using Edit-R EGFP dCas9-VPR mRNA to enrich for …
Source: genprotocols.genengnews.com
Date Published: 7/29/2021
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DharmaFECTトランスフェクション試薬
DharmaFECT Duoは、siRNAとプラスミドDNAを同時に効率よく細胞へ導入するためのトランスフェクション試薬です。 特長; 製品情報; サポートデータ; 関連リンク …
Source: www.horizondiscoverykk.com
Date Published: 3/12/2022
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주제와 관련된 이미지 dharmafect duo protocol
주제와 관련된 더 많은 사진을 참조하십시오 siRNA Transfection Protocol. 댓글에서 더 많은 관련 이미지를 보거나 필요한 경우 더 많은 관련 기사를 볼 수 있습니다.
주제에 대한 기사 평가 dharmafect duo protocol
- Author: Thermo Fisher Scientific
- Views: 조회수 85,273회
- Likes: 좋아요 334개
- Date Published: 2012. 8. 7.
- Video Url link: https://www.youtube.com/watch?v=P-TVDe4OrpM
What is DharmaFECT?
DharmaFECT 1 is the most all-purpose transfection reagent, demonstrating efficient, low-toxicity delivery to over 80% of validated cell types.
How much siRNA do you use for transfection?
In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point).
How do transfection reagents work?
Transfection reagents, such as Transfectamine™ 5000, are positively charged and attract the negatively charged DNA to form a positively charged polymer, which can interact with negatively charged cell membrane which enables the uptake of this polymer into the cell.
What is lipofectamine transfection?
Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.
Can I resuspend siRNA in water?
SiRNA buffer is recommended for long term storage, you can resuspend your SiRNA in RNAse free molecular grade water (or 0.22µm filtered DEPC treated water) for immediate use.
How long can siRNA last?
Gene silencing resulting from siRNA can be assessed as early as 24 hours post-transfection. The effect most often will last from 5–7 days. However, the duration and level of knockdown are dependent on the cell type and concentration of siRNA. Transfections may be repeated to maintain silencing.
How do you mix siRNA?
To dilute the 5x siRNA Buffer to 1x siRNA Buffer, mix four volumes of sterile RNase-free water with one volume of 5x siRNA Buffer. The composition of the 1x siRNA Buffer is 60 mM KCl, 6 mM HEPES-pH 7.5, and 0.2 mM MgCl2.
Can I Vortex siRNA?
Incubate the diluted siRNA mixture at room temperature for 5 min. Important: do NOT vortex the diluted siRNA mixture.
What is difference between siRNA and shRNA?
Definition. siRNA refers to a single-stranded RNA molecule produced by the cleavage and processing of double-stranded RNA while shRNA refers to a short sequence of RNA which makes a tight hairpin turn and can be used to silence gene expression. Thus, this is the main difference between siRNA and shRNA.
How do you get siRNA into cells?
After entering the tissue interstitium, siRNA is transported across the interstitial space to the target cells. After reaching the target cell, siRNA undergoes internalization via endocytosis, a process that involves siRNA being encapsulated in endocytic vesicles that fuse with endosomes.
How is siRNA concentration calculated?
What is your target concentration to treat the cells? Like, if you want to treat 100 nM concentration, the calculation will be ((100 nM/20 uM)*500 uL) = ((100 nM/20 x1000 nM)*500 uL) = 2.5 uL (of stock siRNA).
How do you find the transfection efficiency of siRNA?
You can try FISH to detect it in the cells but basically any molecular beacon that binds your RNA should work. You can co-transfect a BLOCK-it Fluorescent Oligo from Invitrogen with your siRNA as an indicator of transfection efficiency.
How do you treat cells with siRNA?
- Treat the cells with siRNA.
- Wait for 4-12 hours and change the media (depending on cell line, you may wanna optimize it first by checking the expression in 48. …
- Wait for 48 hours (from the treatment) and introduce your drug.
- Wait for 24* hours (from the introduction of the drug) and do your analyses.
How does siRNA affect gene expression?
The siRNA-induced post transcriptional gene silencing starts with the assembly of the RNA-induced silencing complex (RISC). The complex silences certain gene expression by cleaving the mRNA molecules coding the target genes.
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Optimizing siRNA Transfection for RNAi
Efficient Transfection is Critical
The ability of small interfering RNAs (siRNAs) to silence gene expression is proving to be invaluable for studying gene function in cultured mammalian cells. Quite often, the success or failure of an siRNA experiment hinges on siRNA delivery. siRNAs can be transiently transfected using commonly available transfection reagents. However, high efficiency transfection of siRNA is not trivial; therefore, the utility of RNAi in difficult-to-transfect cell types may be limited.
To achieve maximum effectiveness of exogenously introduced siRNAs, transfection optimization experiments are required. Failure to optimize critical transfection parameters can render RNAi effects undetectable in cell culture. These transfection parameters include culture conditions, choice and amount of transfection agent, exposure time of transfection agent to cells, and siRNA quantity and quality. The transfection procedure itself can be a critical factor. The pre-plated transfection procedure involves pre-plating cells, meaning the cells are allowed to attach, recover, and grow for 24 hours prior to transfection. Here, we show evidence that an alternative transfection procedure, termed reverse transfection [1] or neofection [2], offers several key benefits over the traditional pre-plating method. Reverse transfection involves simultaneously transfecting and plating cells, much like procedures used for transfecting suspension cells. This method is easier and faster because it bypasses several steps of the traditional procedure. This article summarizes the use of reverse transfection to maximize performance of siRNA in cultured cells and offers suggestions on how to optimize siRNA transfection parameters.
Important Parameters in siRNA Transfection Experiments
Health of cultured cells
Transfection method
Transfection conditions
Quality and quantity of siRNA
Figure 1. Standard Pre-Plated Transfection vs. Reverse Transfection with siPORT™ NeoFX™ Transfection Agent. Mammalian adherent cells are typically “pre-plated” prior to transfection, allowing them to reattach and resume growth for 24 h before exposure to transfection complexes (left). Reverse transfection or “neofection” involves adding transfection complexes to the cells while they are in suspension, prior to plating, thus saving an entire day in the transfection procedure (right). . Mammalian adherent cells are typically “pre-plated” prior to transfection, allowing them to reattach and resume growth for 24 h before exposure to transfection complexes (left). Reverse transfection or “neofection” involves adding transfection complexes to the cells while they are in suspension, prior to plating, thus saving an entire day in the transfection procedure (right).
Figure 2. Efficient Reverse Transfection of Various Cell Lines. (A) Reverse transfection of a GAPDH siRNA (Silencer GAPDH siRNA, Ambion) into seven different mammalian cell types. Amounts of siPORT™ NeoFX™ (Ambion), siRNA amounts, and cell density were optimized for each cell line (data not shown). All cells were harvested and analyzed by real-time RT-PCR for GAPDH mRNA levels at 48 hours after transfection. (B) HepG2 and HeLa cells were both reverse transfected during plating and transfected after pre-plating the cells with an siRNA targeting GAPDH (Silencer GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion). 48 hours post-transfection, GAPDH expression was measured by real-time RT-PCR. Percent gene expression was calculated as GAPDH gene expression in GAPDH siRNA transfected cells compared to those transfected with the Negative Control siRNA. Reverse transfection of a GAPDH siRNA (Silencer GAPDH siRNA, Ambion) into seven different mammalian cell types. Amounts of siPORT™ NeoFX™ (Ambion), siRNA amounts, and cell density were optimized for each cell line (data not shown). All cells were harvested and analyzed by real-time RT-PCR for GAPDH mRNA levels at 48 hours after transfection.HepG2 and HeLa cells were both reverse transfected during plating and transfected after pre-plating the cells with an siRNA targeting GAPDH (Silencer GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion). 48 hours post-transfection, GAPDH expression was measured by real-time RT-PCR. Percent gene expression was calculated as GAPDH gene expression in GAPDH siRNA transfected cells compared to those transfected with the Negative Control siRNA.
Figure 3. Reverse Transfection Yields Higher Tolerance to Cell Plating Density. (A) SKOv3 cells were reverse transfected in a 96 well plate using 10 nM and 30 nM GAPDH siRNA (Silencer GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion) at the indicated cell densities using siPORT™ NeoFX™ Transfection Agent (0.3 µl per well, Ambion). At 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Remaining gene expression was calculated as a percentage of GAPDH mRNA in cells transfected with GAPDH siRNA compared to cells transfected with Negative Control siRNA. Data were normalized against the 18S rRNA signal. (B) COS-7 cells were pre-plated in a 24 well dish at the indicated plating densities 24 hours prior to transfection. Transfections were performed with either 10 nM GAPDH or Negative Control #1 siRNA using siPORT™ Amine Transfection Agent (4 µl per well, Ambion). Remaining gene expression was determined as described for Panel A. SKOv3 cells were reverse transfected in a 96 well plate using 10 nM and 30 nM GAPDH siRNA (Silencer GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion) at the indicated cell densities using siPORT™ NeoFX™ Transfection Agent (0.3 µl per well, Ambion). At 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Remaining gene expression was calculated as a percentage of GAPDH mRNA in cells transfected with GAPDH siRNA compared to cells transfected with Negative Control siRNA. Data were normalized against the 18S rRNA signal.COS-7 cells were pre-plated in a 24 well dish at the indicated plating densities 24 hours prior to transfection. Transfections were performed with either 10 nM GAPDH or Negative Control #1 siRNA using siPORT™ Amine Transfection Agent (4 µl per well, Ambion). Remaining gene expression was determined as described for Panel A.
Figure 4. Transfection Agent Cytotoxicity. Multiple siPORT™ NeoFX™ Transfection Agent volumes (0.03-1.0 µl, Ambion) were used in reverse transfection of HepG2 cells. Assays were done in 96 well plates with 5 nM GAPDH siRNA (Silencer GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion). Remaining gene expression (bars) was determined as described for Figure 3A. Cell viability (line) was also measured using the ViaCount Assay (Guava Technologies). As reagent volume increased, greater levels of siRNA-mediated reduction of target gene expression were obtained. In this cell type, siPORT NeoFX shows minimal toxicity and yields a broad range of silencing activity. Some reagents are not as flexible and require more precision. volumes (0.03-1.0 µl, Ambion) were used in reverse transfection of HepG2 cells. Assays were done in 96 well plates with 5 nM GAPDH siRNA (Silencer GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion). Remaining gene expression (bars) was determined as described for Figure 3A. Cell viability (line) was also measured using the ViaCount Assay (Guava Technologies). As reagent volume increased, greater levels of siRNA-mediated reduction of target gene expression were obtained. In this cell type, siPORT NeoFX shows minimal toxicity and yields a broad range of silencing activity. Some reagents are not as flexible and require more precision.
Figure 5. Exposure Time to Transfection Complexes. HeLa cells (5 x 103 cells/well) were exposed to transfection complexes containing one of two concentrations of transfection agent (1-2 µl) + 10 nM GAPDH (Silencer GAPDH siRNA, Ambion) or negative control siRNA (Silencer Negative Control #1 siRNA, Ambion). Medium was changed to remove transfection complexes at different time points. Cellular viability, apoptosis, and siRNA activity were measured 48 h after transfection began. Cell viability (blue line) was measured using the ViaCount Assay (Guava Technologies). Apoptosis (yellow line) was measured using a Guava PCA™-96 instrument (Guava Technologies). Remaining gene expression (green bars) was determined as described for Figure 3A. NT = Not Transfected HeLa cells (5 x 103 cells/well) were exposed to transfection complexes containing one of two concentrations of transfection agent (1-2 µl) + 10 nM GAPDH (Silencer GAPDH siRNA, Ambion) or negative control siRNA (Silencer Negative Control #1 siRNA, Ambion). Medium was changed to remove transfection complexes at different time points. Cellular viability, apoptosis, and siRNA activity were measured 48 h after transfection began. Cell viability (blue line) was measured using the ViaCount Assay (Guava Technologies). Apoptosis (yellow line) was measured using a Guava PCA™-96 instrument (Guava Technologies). Remaining gene expression (green bars) was determined as described for Figure 3A. NT = Not Transfected
Figure 6. Optimal Amount of siRNA. HeLa cells were split and resuspended in growth media at 4.0×104 cells/ml. Transfection complexes were prepared containing the indicated concentration of chemically synthesized GAPDH siRNA (Ambion) or Negative Control siRNA #1 (Ambion; data not shown), and 0.3 µl siPORT™ NeoFX™ Transfection Agent (Ambion). 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Remaining gene expression was determined as described for Figure 3A. HeLa cells were split and resuspended in growth media at 4.0×104 cells/ml. Transfection complexes were prepared containing the indicated concentration of chemically synthesized GAPDH siRNA (Ambion) or Negative Control siRNA #1 (Ambion; data not shown), and 0.3 µl siPORT™ NeoFX™ Transfection Agent (Ambion). 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Remaining gene expression was determined as described for Figure 3A.
The goal of transfection optimization is to determine the conditions that will provide maximum gene knockdown while maintaining an acceptable level of viability for the particular cell type (see sidebar, Two-Step Optimization Protocol).For maximal cell viability during transfection, cells must be healthy at the beginning of the experiment–healthy cells are easier to transfect than poorly maintained cells. Overly crowded and sparse cultures are not conducive for cell health. Many cells undergo expression profile changes that can adversely affect your experiments when they are stressed by culture conditions. As a rule, cells should never be allowed to cover the entire surface area of their culture dish. Instead, cells should take up between 20 and 80 percent of the available space. Subculturing cells before they become overcrowded minimizes instability in continuous cell lines and reduces variability from experiment to experiment. Cells can gradually change in culture, and it is difficult to consistently maintain cells in perfect health; therefore, to obtain maximally reproducible experimental results, we recommend that cells be transfected within 10 passages of the optimization experiments. Cells older than this should be destroyed and replaced with new cells from a frozen stock. Finally, maintaining strict protocols, including time intervals between plating and transfecting cells, will improve experimental reproducibility.In preparation for transfection, adherent mammalian cells have been traditionally pre-plated into tissue culture wells and allowed to attach, recover, and grow for 24 h prior to transfection. Reverse transfection is an alternative method of transfection where cells are transfected while still in suspension (i.e. after trypsinization and prior to plating). The method produces equivalent or improved transfection efficiency over the standard pre-plated method for many of the cell types tested and saves an entire day in the process (Figure 1). Presumably, the amount of exposed cell surface, and not the number of transfection complexes, is the limiting factor in traditional adherent transfection. Reverse transfection is believed to increase cell exposure to transfection complexes often leading to greater transfection efficiency.Figure 2A shows reverse transfection of a GAPDH siRNA into seven different mammalian cell types. The data suggests that reverse transfection can deliver high levels of functional siRNA to a wide variety of cells. Some cell types transfected more efficiently by reverse transfection than the traditional method. For example, HepG2 cells, traditionally a difficult cell line to transfect, reverse transfected remarkably well (Figure 2B), perhaps because their dense growth pattern precludes adequate cell surface exposure to transfection agents once attached to a substrate.Cell density became a less critical parameter, requiring little to no optimization, when cells were reverse transfected. Figure 3A demonstrates that a broad range of cell concentrations were reverse transfected efficiently, whereas traditional pre-plated transfections required careful optimization of cell density (Figure 3B). In addition, reverse transfection is faster–a full day can be saved because cells do not have to be plated prior to transfection. Because of these fundamental advantages, Ambion scientists routinely optimize transfection of new cell lines using the reverse transfection procedure.Overall, transfection efficiency and cell viability are dependent on choice and amount of transfection agent and exposure time of cells to transfection agent. Commercially available reagents perform with varying levels of effectiveness depending on the cell type. A successful match between cell line and reagent can usually be made by testing several commercially available agents. Transfection agent volume is also critical–too little will not transfect efficiently; too much can be cytotoxic. Both siRNA transfection efficiency and cell viability should be considered when designing transfection agent screening experiments. The ideal reagent is one that yields effective target gene reduction without significant cell mortality. In HepG2 cells, siPORT™ NeoFX™ Transfection Agent shows minimal toxicity and yields a broad range of silencing activity (Figure 4). Some reagents and cell lines are not as flexible and require more precision. Ambion recommends testing transfection agents that have been validated specifically for siRNA transfection. We have found that most DNA-based transfection agents are ineffective for siRNA delivery.Length of cell exposure to transfection agents should be optimized to minimize cellular toxicity and maximize siRNA activity by varying the amount of transfection agent and cell exposure time to transfection complexes (Figure 5). Media containing transfection agent was removed from the wells at the indicated time points and replaced with fresh media. Cellular viability, apoptosis, and siRNA activity were measured 48 hours after addition of 1 or 2 µl transfection agent + GAPDH siRNA or negative control siRNA. In wells where transfection agent was not removed, cells appeared necrotic, they underwent moderate levels of apoptosis, and cell viability was >30% less than a nontreated control well. These same samples, however, showed >90% reduction in GAPDH gene expression over negative control wells. When transfection complexes were removed at 4 hours post transfection, cell viability was >95%, apoptosis was minimal, but GAPDH silencing was 30% less than in cultures experiencing no media change. When cells were exposed to 1 µl transfection agent for 24 hours post-transfection before a media change, GAPDH silencing was high, comparable to cells with no media change, and cell viability was nearly 90%. These data suggest that careful optimization of cell exposure to transfection complexes can improve the quality of data generated in RNAi experiments.The quality and quantity of siRNA used for transfection significantly influences RNAi experiments. siRNA should be free of contaminants carried over from synthesis including salts, proteins, and ethanol. Additionally, the siRNA should also be <30 bp, because the presence of dsRNA larger than approximately 30 bp has been shown to alter gene expression by activating the nonspecific interferon response [3].The optimal concentration of siRNA is influenced by several factors including properties of the target gene and cell type. As mentioned above, too much siRNA may lead to off-target effects; too little can result in undetectable gene silencing. In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point). In Figure 6, transfection of HeLa cells was optimized at very low concentrations of siRNA. HeLa cells are easier to transfect than many other cell types, and 10 nM siRNA in combination with reverse transfection is sufficient for obtaining optimal target gene reduction.
GEN Protocols
Procedure
General protocol for lipid transfection of Edit-R Fluorescent dCas9-VPR mRNA and synthetic guide RNAs and enrichment of transfected cells
The following is a general protocol using Edit-R EGFP dCas9-VPR mRNA to enrich for transfected cells using fluorescence activated cell sorting (FACS). Exact reagent amounts and parameters for both lipid-mediated transfection and electroporation should be empirically determined through careful optimization in cells of interest prior to experimentation. The protocol below describes delivery conditions in U2OS cells using the DharmaFECT Duo transfection reagent and is given for illustrative purposes only.
All steps of the protocol should be performed in a laminar flow cell culture hood using sterile technique.
Day 1
Trypsinize and count cells. Plate cells in 6-well plates using growth medium at a cell density so that the cells are 70 to 90% confluent the next day. For example, U2OS cells should be diluted to 100,000 cells in 1 mL of medium for plating at 250,000 cells/well in a 6-well plate. Incubate cells at 37 °C with 5% CO2 overnight.
Day 2
Prepare a 100 ng/µL EGFP dCas9-VPR mRNA working solution by thawing EGFP dCas9-VPR mRNA on ice and adding 20 µL of 1 µg/µL stock solution of fluorescent Cas9 mRNA to 180 µL of Tris buffer. Verify the EGFP dCas9-VPR mRNA concentration using UV spectrophotometry at 260 nm and adjust the volume if necessary to obtain 100 ng/µL. Prepare guide RNA reagents for transfection. For crRNA and tracrRNA: a. Prepare a 10 µM crRNA stock solution by adding the appropriate volume of Tris buffer to crRNA. Verify the RNA concentration using UV spectrophotometry at 260 nm and adjust the volume if necessary to obtain 10 µM.
b. Prepare a 10 µM tracrRNA stock solution by adding the appropriate volume of Tris buffer to tracrRNA. Verify the RNA concentration using UV spectrophotometry at 260 nm and adjust the volume if necessary to obtain 10 µM.
c. Prepare a 2.5 μM crRNA:tracrRNA transfection complex by adding 25 μL of crRNA and 25 μL of tracrRNA to 50 μL of Tris buffer (total volume is 100 μL).
For synthetic sgRNA:
For synthetic sgRNA: a. Prepare a 2.5 µM synthetic sgRNA stock solution by adding the appropriate volume of Tris buffer to the sgRNA. Verify the RNA concentration using UV spectrophotometry at 260 nm and adjust the volume if necessary to obtain 2.5 µM. In a 15 mL conical prepare for each sample to be transfected as described in Table 2 (columns 2-4) for a final 25 nM concentration of the guide RNA and 5 µg/well of EGFP dCas9-VPR mRNA. In a separate tube, prepare a 30 μg/mL DharmaFECT Duo working solution by diluting 30 μL of 1 mg/mL stock DharmaFECT Duo transfection reagent in 1 mL serum-free medium and mix gently; this volume is sufficient for 4 wells with 7.5 μL/well in 6-well format. Incubate for 5 minutes at room temperature.
5. Add 250 μL DharmaFECT Duo working solution to each sample tube as shown in Table 2 (column 5); this will result in 3 μg/well final concentration. DO NOT add DharmaFECT Duo working solution to the untransfected control, which should contain serum-free medium only. This brings the total volume to 500 μL in each tube. Mix by pipetting gently up and down and incubate for 20 minutes at room temperature.
6. Prepare transfection medium by adding 2,000 μL antibiotic-free complete medium to each sample to bring the total volume in each tube to 2,500 μL (columns 6 and 7).
7. Remove medium from the wells of the 6-well plate containing cells and replace with 2,500 μL of the appropriate transfection medium to each well.
Day 3
8. After 24 hours, trypsinize cells. Collect ¾ of the cells leaving ¼ as a presorted population.
9. Centrifuge and wash cell pellet with PBS to remove medium. Centrifuge again and resuspend cells in appropriate cell sorting buffer
Expression of fluorescent protein over time should be examined for your cells to determine the optimal time to sort for enrichment. We suggest a minimum of 24 hours after lipid transfection to allow for translation of the mRNA into dCas9-VPR and fluorescent proteins, and 8-24 hours for electroporation. Cell sorting will need to be performed before turnover of the fluorescent protein, which will be dependent on the half-life in your experimental cells.
For optimal enrichment, we suggest collecting cells with high fluorescence intensity, selecting the top 10% of fluorescent cells.
Clonal cell lines can be created through sorting single cells into individual wells of a 96-well plate.
11. Expand cell populations in new plates with an appropriate well size corresponding to the number of cells collected.
12. Incubate cells at 37 °C with 5% CO2 for an additional 48 hours before proceeding with gene editing analysis.
Gene activation assay recommendations
The most commonly used method for detection of editing events in a cell population is RT-qPCR. This assay can be performed using a standard cDNA synthesis kit (ThermoFisher, Cat #K1641) and RT-qPCR protocol (Applied Biosystems TaqMan Assay).
Figure 1.Representative FACS data of the untransfected sample (top) and transfected sample containing Edit-R EGFP dCas9-VPR mRNA (bottom). The untransfected sample should be used to define which cells are negative (Neg) for fluorescence and to be excluded from sorting. Gates are drawn around the dim and top 10% fluorescent populations to be sorted and collected.
DharmaFECT transfection reagents
Editing of PSMD7 gene in U2OS-(Ubi)EGFP cells using Edit-R Cas9 Nuclease protein NLS delivered by DharmaFECT transfection reagents
U2OS-(Ubi)EGFP cells were plated at 10,000 cells/well in 96-well plates and co-transfected using DharmaFECT transfection reagents with 25 nM Edit-R Cas9 Nuclease protein NLS and synthetic crRNA:tracrRNA at 50 or 100 nM targeting PSMD7. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. UT = untreated sample, MW = FastRuler Low Range DNA Ladder (Thermo Scientific).
Editing of VEGFA gene in HEK293T cells using Edit-R Cas9 Nuclease mRNA delivered by DharmaFECT transfection reagents
HeLa293T cells were plated at 20,000 cells/well in 96-well plates and co-transfected using DharmaFECT transfection reagents with 200 ng of Edit-R-Cas9 mRNA and synthetic crRNA:tracrRNA targeting VEGFA. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. DF1 = DharmaFECT 1, Duo = DharmaFECT Duo, UT = untreated sample, MW = FastRuler Low Range DNA Ladder (Thermo Scientific).
DharmaFECT reagents provide an improved dynamic range
DharmaFECT reagents are effective across a broader range of experimental conditions when compared to Lipofectamine® 2000 (Invitrogen). Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Three cell densities of HepG2 cells were transfected with GAPD siRNA (100nM) using a range of volumes (0.05 to 1.6μL/well) of Lipofectamine 2000 and DharmaFECT 4 transfection reagents. mRNA levels (bars) were assessed by branched DNA assay (Panomics Quantigene® Reagent System) and cell viability (data points) was determined by alamarBlue® (Biosource International).
DharmaFECT Transfection Reagents provide highly efficient delivery at low siRNA concentrations
To determine transfection efficiencies at low siRNA concentrations, two genes were targeted with various amounts of SMARTpool siRNA reagent. DharmaFECT achieved > 80% silencing at all siRNA concentrations, while HiPerFect (Qiagen) only achieved this level with PPIB at 100 nM. HeLa cells were transfected with SMARTpool TM reagents targeting Cyclophilin B (PPIB) or MAPKI at concentrations of 1, 5, 25, and 100 nM. mRNA expression (bars) was determined by branched DNA assay (Panomics Quantigene® Reagent System) and cell viability (data points) was determined by alamarBlue® (Biosource International).
DharmaFECT outperforms other reagents in optimization study
Table adapted from Borawski et al. A study by scientists at Novartis concluded that in transfection optimization in 384-well format (in preparation for screening) one of the four DharmaFECT transfection reagents outperformed all other reagents in delivery efficiency and overall cell viability in 9 of 10 cell lines. Lipid transfection reagents tested were DharmaFECT 1-4, HiPerFect® (Qiagen), TransIT-TKO® (Mirus) Lipofectamine® 2000 and Oligofectamine® (Invitrogen). J. Borawski et al., Optimization Procedure for small interfering RNA Transfection in a 384-well format. J. Bimolecular Screening,12, 546-559 (2007).
키워드에 대한 정보 dharmafect duo protocol
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사람들이 주제에 대해 자주 검색하는 키워드 siRNA Transfection Protocol
- sirna transfection
- sirna protocol
- sirna transfection protocol
- transfection sirna
- sirna transfection reagent
- sirna transfection efficiency
- transfection of sirna
- rnai transfection
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