Tips for DNA transfection
Cells
Do not use freshly thawed cells. After thawing and before transfection, passage your cells at least 3 times in order to give them time to recover and return to their normal behaviour.
Passage your cells when they are in log-phase growth and before they reach confluency.
Plate your cells the day before transfection in order to reach 60-80% of visual confluency on the day of transfection.
Cells should be healthy and actively dividing, they should not be in culture for too long (> 8 weeks). Cell viability can be assessed using OZ Biosciences' WST-8 Cell Proliferation Kit or MTT Kit (#MT01000) and senescence can be checked with OZ Biosciences' senescence kit (#GXS003).
Cells and media must be free of contamination (mycoplasma, yeast, bacteria).
Cells should be maintained in optimal culture conditions.
DNA Quality
DNA solution must be prepared in DNase/RNase free water or TE buffer.
DNA must be as pure as possible, free of contaminants (proteins, lipids, carbohydrates) and endotoxins.
Abs 260/280nm of the DNA solution should be between 1.7 and 1.9. Do not use DNA solution with lower or higher ratio for transfection.
Ensure that your promoter is compatible with the cells to be transfected.
Ratio of Transfection Reagent
It's critical to test multiple reagent:DNA ratios in order to find the right ratio that achieves the highest transfection efficiency with minimal toxicity. Optimize the reagent/DNA ratio by using a fixed amount of DNA (µg) and varying the volume of the transfection reagent. For example, vary the concentration of Helix-IN Transfection Reagent from 1–2.5 μl per 1 μg DNA to find the optimal ratio.
DNA Amount
It is important to refer to the specific protocol of the reagent you are currently using and to keep in mind that all cells doesn't require the same quantity of DNA.
Use different quantities of DNA as suggested in the instruction manual/related protocol with the optimized tranfection reagent:DNA ratio. You may adjust the amount of DNA depending on the transfected cell lines and the transfection reagent.
Be sure to keep DNA:Reagent ratio constant when adjusting DNA dose.
Controls
Positive controls are highly recommended for your transfection experiments. OZ Biosciences offers a large range of pVectOZ control plasmids encoding the most common reporter genes (GFP, b-Gal, Luciferase, SEAP, CAT).
Mock transfection: perform a transfection control without any DNA so that eventual non-specific effects due to the transfection reagent can be observed.
Transfection practices
We recommend preparing the DNA/reagent complexes in DMEM or PBS without any supplement.
Allow the reagent to reach room temperature before starting your experiment.
Once DNA is added to the transfection reagent, incubate for 20 min at room temperature for optimal complexes formation and add directly the mix onto your cells. Do not wait for more than 30 minutes once the DNA/reagent complexes are formed.
When using Helix-IN do not incubate complexes Helix-IN/DNA less than 30 min at RT
Distribute the complexes onto the cells in a dropwise manner and gently rock the plate to ensure even dispersion.
N/P Charge Ratios
Numerous research studies reported the importance of the charge ratio between the positive cationic lipids and the negative DNA to generate lipoplexes for successful plasmid delivery. These assessments concluded to a considerable effect of the charge ratio on physicochemical features of the lipoplexes and subsequently on the transfection efficiency. Usually, increasing the charge ratio leads to improving electrostatic interaction between the cationic lipid and the DNA. Nevertheless, above the optimal charge ratio it has been found that a dose-dependent increase of cytotoxicity occurs. This charge ratio is known at the N/P ratio, calculated based on the positive charge from the amino groups in the cationic lipids (N) and the phosphate group of the DNA backbone (P) (or the negative charges of the amino acids in proteins) concentration. Herein, DOTAP has one nitrogen carrying one positive charge. As a direct consequence, 1 nmol of DOTAP contribute to 1 nmol of positive charge. On the other hand, 1 µg of DNA contributes to 3.1 nmol of negatively charged phosphate.[1] Mohammed-Saeid, W., et al. Di-peptide-modified gemini surfactants as gene delivery vectors: exploring the role of the alkyl tail in their physicochemical behavior and biological activity. The AAPS journal, 2016, 18, 1168-1181. [2] Datta, D., et al. Impact of the Charge Ratio on the In Vivo Immunogenicity of Lipoplexes. Pharmaceutical Research, 2017, 34, 1796-1804.
Tips for siRNA transfection
Cells
Do not use freshly thawed cells. After thawing and before transfection, passage your cells at least 3 times in order to give them time to recover and return to their normal behaviour.
Passage your cells when they are in log-phase growth and before they reach confluency.
Plate your cells the day before transfection in order to reach 60-80% of visual confluency on the day of transfection.
Cells should be healthy and actively dividing, they should not be in culture for too long (> 8 weeks). Cell viability can be assessed using OZ Biosciences' MTT Kit (#MT01000) and senescence can be checked with OZ Biosciences' senescence kit (#GXS003).
Cells and media must be free of contamination (mycoplasma, yeast, bacteria).
Cells should be maintained in optimal culture conditions.
siRNA
Use RNase-free materials.
Use high quality siRNA (PAGE purified and desalted).
If possible, work with low siRNA concentrations (1-10nM) in order to avoid off-target and/or cytotoxic effects.
Do not use water to diulte your siRNA stock solution, prefer the manufacturer's buffer or 100mM NaCl in 50mM Tris (pH 7.5) to avoid denaturation.
Calculate the correct amount of siRNA: the molecular weight of an average siRNA molecule is approx. 13.500.
Controls
Positive control: use siRNA directed against a housekeeping gene (i.e. GAPDH) or labeled siRNA.
Negative control: use mismatch and/or non-targeting sequence as negative control.
Transfection practices
We recommend preparing the siRNA/reagent complexes in DMEM or PBS without any supplement.
Allow the reagent to reach room temperature.
Once siRNA is added to the transfection reagent, incubate for 20 minutes at room temperature for optimal complexes formation and add directly the mis onto your cells. Do not wait for longer than 30 min once the complexes are formed.
Distribute the complexes onto the cells in a dropwise manner and gently rock the plate to ensure an even dispersion.
Optimal silencing after siRNA transfection can be obtained after 24h for mRNA expression assay up to 72h for protein expression assay.
What are the main differences between Lipofection and Polyfection?
Lipofection and polyfection (respectively lipid-based and polymer-based transfection) are two methods of transfection using synthetic vectors to deliver nucleic acids into cells.
Even if the finalities of the two techniques are the same, some differences still exist orienting the nucleic acid delivery applications to one or the other (refer to table below).
The principal difference between the two technologies comes from the capacity to “easily” modify, graft, branch or change amount of amines or hydrophobicity of polymers; this results in highly condensing and compacting DNA for a more efficient delivery into nucleus. Lipofection main advantage relies on its versatility: all nucleic acids and even proteins can be delivered into cells as opposed to Polyfection that is preferentially dedicated to DNA applications.
The choice of the method for transfection will depend on the cell type and the application.
Lipid-based reagents are less recommended if lipid signaling is studied when polyfection will not be advised for transfecting suspension cell lines. Both techniques are compatible with stable cell lines generation and should be approached sparingly when dealing with primary cells: in this case, Magnetofection must be highly considered.
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Magnetofection Generalities
Do starting kits contain all the necessary materials for customers to make their experiment ?
All the starting kits contain the necessary material for Magnetofection-based experiments. Let’s take the KC30200 Starting Kit for example. This kit contains: A super magnetic plate (MF10000) + 100µL each of PolyMag (PN30100), CombiMag (CM20100) + PolyMag Neo (PG60100). Depending on the kits, samples and volumes may vary.
Can I use the SUPER MAGNETIC PLATE (ref# MF10000) with wells, dishes and flasks? When should I use the Magnetic plate MF10096 or the Mega Magnetic plate MF14000?
The super magnetic plate MF10000 is designed to fit any culture plate from a 384-well plate to a 6-well plate. Its standard format is suitable for any cell culture model from a single 35 mm dish, to a 75cm² flask. The size corresponds to a cell culture plate (8.3cmx12.5cm) and thus is suitable for one plate at a time. This is the one we recommend for testing the Magnetofection products and that we send to customers for reagents evaluation. - The MF10096 has been specifically designed for 96 well plates. Each magnet is directly underneath each well of the culture plate so that a strong magnetic field is directed in each well. - The MF14000 , Mega Magnetic Plate, is similar to the Super Magnetic Plate but bigger (20.1cmx25.7cm), and thus is suitable for holding up to 4 plates at one time. Its surface corresponds to 4 Super magnetic plates (MF10000).
How long iron nanoparticles stay within the cells after transfection ?
All of our nanoparticles are totally biodegradable and they won’t interfere with the cell's biology: the iron core and their coating are made of specific material allowing degradation and biocompatibility in cells, tissue and organisms. Whereas in whole organism, iron oxides have been shown to quickly degrade through natural iron metabolism pathways (iron ions are incorporated into the hemoglobin pool), iron beads will last a little more longer in cells: this period may vary from one week to 3 weeks depending the nanoparticle doses. Nevertheless, unless pathway to study implies iron metabolism, there won’t be any interference in cell signaling while iron nanoparticles are present in cells. It is important to consider that nanoparticles penetrate into cells through endocytosis; there isn’t any physical action that could damage the cell membrane. Moreover, on a practical note, magnetic nanoparticles won’t interfere either with any molecular biology experiments. From DNA or RNA analysis (PCR, qRT-PCR) to protein experiments (protein quantification, WB, Immunoblots, enzyme measurement) all the standard procedures can be performed
How many nanoparticles would go inside a typical cell type (say HEK-293, NIH-3T3, human fibroblasts) on a single transfection ?
We don’t really know the number of nanoparticles that go inside the cells during a Magnetofection procedure. Nevertheless we can give you some indications that can be used to calculate the number of nanoparticules per cell (in theory, but this will depend on the volume of reagent used and the cell culture format): The number of nanoparticles is estimated around 2.5 x 10e9 up to 2 x 10e12 particles per mL. This is just an indication because informations regarding the concentration and formulation of the reagents are confidential.
How long does it take to degrade iron nanoparticles ?
The degradation time will depend on the cell model (particularly its iron metabolism), the iron content of the nanoparticles, etc. We can estimate that the nanobeads stay for 3 or 4 days inside the cells before the beginning of degradation. Again, this time value for degradation will depend on the cells (in PBMC, 15 days after Magnetofection, some cells are still attracted by external magnets) and on other factors. The major point here is that our nanoparticles are totally biodegradable and compatible even for in vivo experiments (In vivo, the degradation will follow the pathway of iron metabolism).
Can Magnetofection be used for reverse transfection ?
Magnetofection can be used in reverse transfection. The protocol is as simple as the “classic” Magnetofection : Follow the protocol for the complex formation. Once they are formed, add the complexes (DNA/nanoparticles) to the dish. Then, apply the magnetic field to attract them down for at least 5 to 10 min. Finally, keep the culture plate onto the magnetic plate and add your cells directly into wells containing the complexes. Then, follow the standard protocol until evaluation of the treansgene expression.
What would be the effect of leaving the transfected plate on the magnet for a long time (24/48 hours?)
Long term incubation of the cell culture dish onto the magnetic plate will have no effect. The reason we are recommending to incubate the cells onto the magnetic plate for 20 minutes is that the plateau (of NeuroMag/DNA complexes uptake for example) is reached after 10-15 minutes. Leaving the cells onto the plate longer will not increase the efficiency.
LipoMag Kit / MagnetoFectamine
How does Magnetofectamine (Lipofectamine 2000 + CombiMag) compare to LipoMag Kit (DreamFect Gold + CombiMag)?
It is a difficult question because efficiency is highly variable from cells to cells and from cell culture conditions to cell culture conditions. Meaning that one lab can prefer LipoMag Kit (CombiMag + DreamFect Gold) and another lab can prefer Magnetofectamine even if they are working on the same cells. The best would be to test both kits and compare; otherwise our recommendation will be based essentially on the kind of cells you want to transfect. If you're unsure which one to test, please do not hesitate to contact us for advice.
Do you have an alternative to Magnetofectamine for lentivirus production in HEK ?
These cells are very easy to transfect and thus, Magnetofection may not be the only way to get a high yield of lentiviruses production. Actually, Magnetofection is generally proposed for primary cells or hard-to-transfect cells. That’s why we would recommend our DreamFect Gold transfection reagent that is really powerful and can be used for LV production. This reagent allows multiple plasmid co-transfections. So we would suggest you to try producing LV with DreamFect Gold (DG) in parallel to Magnetofectamine.
NeuroMag
Can we use NeuroMag on cells growing on glass ?
Definitively yes; one of the first work describing the use of Magnetofection in neurons was performed onto glass coverslips. You can refer to Buerli et al, 2007, Nature Protocols for more information (see our citation database). In our lab, we routinely perform transfection with NeuroMag on cells growing on glass without any loss of efficiency.
We consistently get transfected astrocytes using the NeuroMag reagent. What should I do?
We’ve already observed that NeuroMag preferentially transfect astrocytes or glial cells when the DNA quantity or the NeuroMag ratio was not optimized to the experimental model. We would recommend to try keeping unchanged the DNA quantity and to vary the NeuroMag volume: for example, use 1 µg DNA with either 1, 2 or 3 µL NeuroMag. Depending on the cell model, the DIV, the medium used, ratio has to be optimized for each lab. Our technical team will be pleased to help you set up the optimal conditions.
In your NeuroMag protocol, what does "Add the NeuroMag / DNA complexes onto cells [growing in culture feeding medium if > 10 DIV or culture medium if < 10 DIV]" means ?
Cell culture conditions should be changed depending on the DIV: - For long-term cultures (>DIV 10), it is recommended to change 50% of the medium every 3 days from DIV 5 (so the first medium change is at DIV 8). Moreover, a higher initial cell density is necessary (800,000 cells per 35 mm dish). 24 hours before transfection, replace 50% of the culture medium with fresh medium. Be sure that your cell cultures don’t become activated before the transfection experiment: a mechanical shock, an electrical activation, a medium change in the hour before the experiment may lead to glial cells activation, ending with a high level of transfected glial cell instead of neurons.
PolyMag / PolyMag Neo
What is the difference between the PolyMag and PolyMag Neo ?
If the basic characteristics of the two reagents are quite similar, their behaviours are totally different. PolyMag Neo is an improved version of PolyMag. PolyMag Neo is more efficient than PolyMag in many cell models in term of % of transfected cells and transgene expression level. This is due to its high capacity of DNA compaction compared to PolyMag. Thus, as PolyMag Neo is more efficient, less DNA is necessary, otherwise if we use the same DNA quantity than with PolyMag, toxicity may appear ; but it will be only due to the fact that too much DNA are used and enter the cell. In summary PolyMag Neo is a real innovation compared to PolyMag. It allows more DNA to enter into cell, thus lower DNA quantity is needed.
What exactly does "supplement-free medium” mean ?
Supplement free medium means essentially a culture medium without serum, antibiotics, anti-fungic or anti-mycoplasma treatment or non-essential amino acids. Having Glutamine is not a problem; glutamine is not affecting the transfection. In our lab we prefer using OptiMEM or DMEM or even Hepes buffer to form the complexes but RPMI is OK as well.
The beads are made of iron oxide. Can you tell me if they are made of 100% iron oxide? What is the amount of iron in the beads ?
The magnetic nanoparticles are made of iron oxide coated with a specific compound that allows the nanoparticles to interact with nucleic acid and form complexes. This compound plays also an important role inside the cells for releasing the DNA. Thus, our particles are made of iron oxide plus a coating. Overall, iron represents half of the weight of the nanoparticles; the other half is the coating. The exact composition is proprietary and confidential.
SilenceMag
Do you have recommendations for Silence Mag in a 100mm plate ?
We recommend using 20 µL SilenceMag for 10nM siRNA (final concentration) as a starting point. Then, to optimize the conditions, we would recommend: For 1-5 nM siRNA, use 15-25 µL SilenceMag; For 10 nM siRNA, use 20-30 µL SilenceMag; For 20 nM siRNA, use 30-40 µL SilenceMag; for ≥ 50 nM siRNA, use 40-50 µL SilenceMag.
How can I raise gene silencing efficiency or silence gene on a long period of time?
For raising gene silencing efficiency or for silencing gene expression over a long period of time, we generally recommend performing sequential transfections either 24h apart or every three days. The strength of Magnetofection is that you can perform a medium change while keeping cells onto the magnet: magnetic complexes stay attracted onto the cell surface and unbound material is removed. This allows lowering toxicity. In this way sequential transfections can be performed repeatedly. Sequential transfection has been used in many publications to increase gene silencing.
LIPOFECTION
DreamFect Gold
Can Dreamfect Gold withstand multiple freeze-thaw or should I really make small aliquots and store them at -20C ?
DreamFect Gold can definitively withstand multiple freeze-thaw cycles. We generally do not recommend making aliquots of our lipid-based reagents as there may be interactions between plastic tubes and the lipids. Nevertheless, if you want to make some aliquots use polypropylene tubes only.
How to improve efficiency and lower toxicity ?
In order to raise both efficiency and viability, the transfection conditions need to be optimized. The goal is to find the ideal nucleic acid amount and DreamFect Gold (DG) volume. (1) Keep the DG to DNA ratio unchanged (for example 3µL per µg DNA should be ok - i.e. 3:1 ratio) and vary the DNA quantities from 0.125 to 1 µg per well in a 24-well plate. (2) Once the DNA amount is found, vary the ratio from 1:1 to 4:1 or even 5:1 (low amounts of DNA allow using higher DG volumes without increasing toxicity).
DreamFect
How to raise the gene silencing efficiency with DreamFect and siRNA ?
There are 2 options that can be tested: (1) try different culture conditions at the time of transfection, depending on the cells capacities to tolerate this and of course if the following experiments allow it:
a. Transfection in serum free medium: Culture and passage your cells as usual and prepare the complexes of siRNA and DreamFect (DF) as recommended. During incubation of the complexes (20 min at RT), put the cells to be transfected in a serum-free medium. Add the complexes to the cells and 4 hours later add serum to the medium. Some cells are more susceptible to transfection if they are starved.
b. Use different transfection conditions (i.e. changing the volume): Culture and passage your cells as usual. Transfect them in a smaller volume (for instance, use half of the recommended volumes for cell culture and transfection). 4 hours later add medium up to the original volume. This should allow raising the chances of the complexes to meet the cells.
(2)- Optimize the siRNA concentrations; try 25nM to 100 nM with 2 to 4 µL of DF. Lowering the siRNA quantity may improve the final results. siRNA may be seen as a viral attack by the cells and trigger an immune response. Therefore if too much siRNA enters the cells, the result may be the opposite of what is expected.
DreamFect Stem
Can DreamFect Stem also transfect other cells or only stem cells ?
Yes, of course DreamFect stem can transfect other cells but it is optimized for stem cells.
Can you please outline the specific differences between DreamFect, DreamFect Gold and DreamFect Stem ?
DreamFect (DF) is the first generation of our transfection reagent; it is recommended for cell lines.
DreamFect Gold (DG) is the second generation; it is an improved version of DreamFect. Basically, DF and DG should have similar transfection efficiency (ie, number of cells transfected) but DG will lead to higher transgene expression.
DreamFect Stem has been developed specifically for pluripotent stem cells and thus is optimized for these cells (higher efficiency with lower toxicity) whereas DF and DG are more generic for cell lines.
Can DreamFect Stem be used for Mouse Nervous stem cells ?
Even if it’s true that DreamFect Stem was designed for any stem cells, we always recommend NeuroMag transfection reagent for Neural Stem Cells. Actually NeuroMag, initially designed for primary hippocampal and cortical neurons has shown a really good efficiency in every “neural cell types”.
Numerous papers have shown its efficiency in primary hippocampal and cortical neurons from any origin as well as cerebellar granules, motor neurons, striatal neurons, astrocytes and even some cell lines. Moreover, we and others have demonstrated that NeuroMag was highly efficient for transfecting Neural Stem Cells without differentiating them; after transfection, toxicity is really low and cells keep their functional characteristics. See for example Pickard M., Biomaterials. 2010; 32(9):2274-84 and Sapet C., Biotechniques. 2011; 50(3):187-9. That’s why we do recommend NeuroMag for this kind of cells. For Neural Stem Cells, DNA quantity should be lowered and ratio to use should be 1:1.
Cells had a tendency to form clumps after adding the mixture of plasmid and DreamFect Stem into cells; toxicity is also observed.
Low efficiency, clump formation and toxicity shouldn’t occur with Dreamfect Stem. This is mainly observed when the transfection conditions are not optimized. Actually it may be necessary to change both DNA amount and the volume of reagent: first vary DNA amount with a fixed ratio of DNA/DreamFect Stem, then vary the ratio of DreamFect Stem with a fixed amount of DNA.
Do I need to change medium the day after transfection? Or can I just leave it on for the next days until evaluation of the experiment ?
It is not necessary to change the medium the day after transfection, but we noticed that a medium change at least 4h after transfection increases the rate of transfected cells and the cell survival.
Lullaby
Lullaby tube was unfortunately placed at -20ºC instead of 4ºC. Is it known if that affects the activity of lullaby significantly ?
Lullaby transfection reagent must be stored at 4°C. However a single stay at -20°C does not impair its capacities.
There is no change in expression from the Q-PCR results 24 and 48 H after siRNA transfection with Lullaby
Gene silencing is generally measured at 72 h after transfection but should be observed when using lullaby after 48H. In order to raise efficiency: (1) optimize the transfection conditions. Depending on many parameters, it may be necessary to try several siRNA concentrations and Lullaby volumes. Actually, some cells will need low amounts of siRNA to shut gene down very efficiently, some other cells will need higher amounts. Thus, we generally recommend trying 5 to 50 nM siRNA with 1 to 4 µL Lullaby in a 24-well plate (volumes has to be adjusted in relation to the plate format). In this way, “ideal” amount of siRNA and Lullaby should be found. (2) Wait 72H. Depending on many parameters (half life of the protein translated from the mRNA to target, the concentration of siRNA delivered into the cells, the immune response of the cell…), it may be necessary to wait until 72H to observe an effect of the silencing. (3) Perform what is called a “reverse transfection”: this means transfecting cells with siRNA at the time of seeding when cells are dissociated.
I would like to test the Lullaby siRNA transfection reagent for co-transfection of siRNA and Plasmids. Is there a special protocol available ?
In order to perform co-transfections (siRNA and DNA), there are two techniques:(1) Use two different reagents: Lullaby (siRNA) and DreamFect Gold (DNA). Form complexes of (siRNA+Lullaby) and (DNA+DreamFect Gold) and after 20 min incubation, mix the two solutions before adding them onto the cells. Even if this protocol worked before for some of our collaborators/customers, it isn’t ideal: some cells may be transfected with both, only with siRNA or only with DNA. (2) Use only DreamFect Gold (DG): DG is a really powerful reagent that presents high compaction capacity allowing co-transfection in an easy way. Simply mix DNA and siRNA together and add them to DG solution. During incubation, complexes will be formed with (siRNA+DNA)+DG. In theory, when plasmid enters the cells, siRNA will also enter since they are in the same complex.
Do you have information about transfecting triphosphate-bearing siRNA ?
Triphosphates bearing siRNA do not interfere with siRNA delivery. On the contrary, they stabilize siRNA and add charges allowing a better interaction with the delivery vehicle. The only recommendation we generally have is for TPP-siRNA produced by in vitro transcription: unbound TPP residues must be carefully removed after transcription to avoid INF response of the transfected cell (Dong-Ho Kim, Nature Biotechnology 22, 321 - 325 (2004)).
Does Lullaby work on cancer cell lines such as MDA-MB-231 ?
Our reagent Lullaby works pretty well with MBA-MB-231 cells (please refer to Monteiro et al., J. Cell. Biol., 2013 and Montagnacet al., Nature, 2013). Usually, the authors transfect cells at low confluence (~20%) with 25 to 50 nM of siRNA.
3D-TRANSFECTION
What would be the advantages of doing this over manipulating cells in 2D before culturing them in 3D ?
There are many advantages of loading 3D-gels with complexes over manipulating cells in classical 2D cultures. For instance:
(1) Cells are transfected in a more “real-life” environment: when cells are cultured in 2D, their behavior (phenotype, metabolic activity…) are totally different than in 3D. So, the reponse to transfection would be totally different in 3D in terms of gene silencing or gene expression. Moreover, most of the commercial reagents that work in 2D don’t work in 3D; meaning that the cells are not in the same state when cultured in/on gel or scaffolds.
(2) If cells are cultured in 2D, they need to be transfected, and let into the cell plate for 24 to 96 hours before gene expression or gene silencing arise. Then, cells are detached with trypsin treatment, washed, counted and loaded into gel. A huge number of manipulations may affect the experiment and what would be observed might not reflect the reality of the silencing or expression.
(3) for in vivo experiments : it is sometime necessary to only inject gel or scaffolds loaded with complexes in order to measure invasion, angiogenesis, bone formation, wound healing…
(4) Cell specificity: Some kinds of cells are very difficult to transfect. For example Neural Stem cells, when cultivated in neuroshperes are really difficult to be transfected. The use of scaffold loaded with complexes have allowed to transfect them in their neurosphere state.
What are the differences between 3D-Fect and 3D-FectIN ?
The goal of these reagents is to form complexes with nucleic acids and once the complexes are formed, they are loaded onto 3D matrices. Because of the huge variety of 3D-supports available, we have developed the 2 reagents :
3D-Fect has been specifically designed for 3D-Scaffold. By 3D-Scaffold, we mean every kind of 3D-matrice that offers a solid support for cell growth. This reagent works from “classic” natural collagen scaffold to synthetic culture insert. This reagent allows forming complexes with nucleic acids, and interacting with 3D surfaces to cover it with the pre-formed complexes. Once the cells colonize the matrices, they come into contact with the complexes and genetic material is delivered while their growing occurs. In this way, cells are transfected directly on the 3D scaffold.
3D-FectIN is designed for 3D-hydrogels such as Matrigel. The mechanism of action is similar to 3D-Fect, it is just a different composition that works better with gels.
Could 3D-Fect and 3D-FectIN be used for mRNA transfection (not siRNA) ?
3D-Fect and 3D-FectIN can definitively be used for mRNA transfection; the protocolsare the same than for DNA: you have to use the same mRNA amount than the DNA amount recommended by the protocol and the same 3D-Fect or 3D-FectIN ratios and volumes.
Tips for DNA transfection
Cells
Do not use freshly thawed cells. After thawing and before transfection, passage your cells at least 3 times in order to give them time to recover and return to their normal behaviour.
Passage your cells when they are in log-phase growth and before they reach confluency.
Plate your cells the day before transfection in order to reach 60-80% of visual confluency on the day of transfection.
Cells should be healthy and actively dividing, they should not be in culture for too long (> 8 weeks). Cell viability can be assessed using OZ Biosciences' MTT Kit (#MT01000) and senescence can be checked with OZ Biosciences' senescence kit (#GXS003).
Cells and media must be free of contamination (mycoplasma, yeast, bacteria).
Cells should be maintained in optimal culture conditions.
DNA
DNA solution must be prepared in DNase/RNase free water or TE buffer.
DNA must be as pure as possible, free of contaminants (proteins, lipids, carbohydrates) and endotoxins.
Abs 260/280nm of the DNA solution should between 1.7 and 1.9. Do not use DNA solution with lower or higher ratio for transfection.
Ensure that your promoter is compatible with the cells to be transfected.
Controls
Positive controls are highly recommended for your transfection experiments. OZ Biosciences offers a large range of pVectOZ control plasmids encoding the most common reporter genes (GFP, b-Gal, Luciferase, SEAP, CAT).
Mock transfection: perform a transfection control without any DNA so that eventual non-specific effects due to the transfection reagent can be observed.
Transfection practices
We recommend preparing the DNA/reagent complexes in DMEM or PBS without any supplement.
Allow the reagent to reach room temperature before starting your experiment.
Once DNA is added to the transfection reagent, incubate for 20 min at room temperature for optimal complexes formation and add directly the mix onto your cells. Do not wait for more than 30 minutes once the DNA/reagent complexes are formed.
Distribute the complexes onto the cells in a dropwise manner and gently rock the plate to ensure even dispersion.
Tips for siRNA transfection
Cells
Do not use freshly thawed cells. After thawing and before transfection, passage your cells at least 3 times in order to give them time to recover and return to their normal behaviour.
Passage your cells when they are in log-phase growth and before they reach confluency.
Plate your cells the day before transfection in order to reach 60-80% of visual confluency on the day of transfection.
Cells should be healthy and actively dividing, they should not be in culture for too long (> 8 weeks). Cell viability can be assessed using OZ Biosciences' MTT Kit (#MT01000) and senescence can be checked with OZ Biosciences' senescence kit (#GXS003).
Cells and media must be free of contamination (mycoplasma, yeast, bacteria).
Cells should be maintained in optimal culture conditions.
siRNA
Use RNase-free materials.
Use high quality siRNA (PAGE purified and desalted).
If possible, work with low siRNA concentrations (1-10nM) in order to avoid off-target and/or cytotoxic effects.
Do not use water to diulte your siRNA stock solution, prefer the manufacturer's buffer or 100mM NaCl in 50mM Tris (pH 7.5) to avoid denaturation.
Calculate the correct amount of siRNA: the molecular weight of an average siRNA molecule is approx. 13.500.
Controls
Positive control: use siRNA directed against a housekeeping gene (i.e. GAPDH) or labeled siRNA.
Negative control: use mismatch and/or non-targeting sequence as negative control.
Transfection practices
We recommend preparing the siRNA/reagent complexes in DMEM or PBS without any supplement.
Allow the reagent to reach room temperature.
Once siRNA is added to the transfection reagent, incubate for 20 minutes at room temperature for optimal complexes formation and add directly the mis onto your cells. Do not wait for longer than 30 min once the complexes are formed.
Distribute the complexes onto the cells in a dropwise manner and gently rock the plate to ensure an even dispersion.
Optimal silencing after siRNA transfection can be obtained after 24h for mRNA expression assay up to 72h for protein expression assay.
Transfection - FAQ
What are the advantages of viral transfection compared to non-viral transfection?
The advantages of viral transduction over non-viral transfection largely depend on the type of virus used. Adenovirus, Retrovirus, Lentivirus, AAV, and others are various types of viruses used as vectors for genetic modification. Despite their structural and/or functional differences, such as the presence or absence of an envelope or specific surface glycoproteins, they have common characteristics that make them superior to non-viral vectors in terms of genetic modification efficiency.
- Higher transduction efficiency: Over time, viruses have evolved to effectively infect cells, and it is on this basis that viral vectors have been developed and optimized for their ability to efficiently transduce host cells by delivering their genetic material. Viral transduction (or genetic modification of cells via viruses) is generally more effective than non-viral transfection methods (lipofection, polyfection, electroporation) because it relies on the intrinsic capabilities of the vectors. For example, Djurovic S et al. conducted a study to determine the optimal gene delivery system in neonatal rat cardiomyocyte cell cultures by comparing the efficiency of gene transfer using adeno-associated viral (AAV) vectors with chemical and physical methods. The transfection efficiency, measured by quantitative chloramphenicol acetyltransferase type I (CAT) assay and β-galactosidase staining based on reporter gene overexpression (LacZ), demonstrated that AAVs were capable of modifying almost 90% of cells without loss of viability; more than 10 times greater than other transfection methods which, in contrast, were toxic to the cells.
- Prolonged and stable expression: Viral vectors have the ability to integrate their genetic material into the host cell genome, which can lead to prolonged and stable expression of the transgene. For example, vectors derived from retroviruses and lentiviruses are known to integrate their viral DNA into the host cell genome, thus ensuring long-term expression of the transgene. Similarly, early in vivo trials using AAVs showed stable expression for several years in mice and up to several months in larger mammals.
- Ability to specifically target certain types of cells: An important feature of viruses is that they exist in a wide range of types and species with varying properties in terms of size, morphology, genetic material, and natural tropism. There are indeed several criteria based on which viruses can be classified. These include the presence of an envelope, symmetry of the viral capsid, nature of viral genetic material (DNA or RNA), virus replication site (nucleus or cytoplasm), and virion size, among others. Additionally, viral vectors can be modified to selectively target certain types of cells by incorporating ligands or specific targeting peptides into their viral envelope. For example, studies have shown that viral vectors can be modified to selectively target cancer cells by incorporating ligands specific to overexpressed receptors. This ability for specific targeting can enhance the efficiency and safety of gene therapies by reducing non-targeted effects.
- Potential for cell reprogramming and gene therapy: Viral vectors are widely used in fields such as cell reprogramming and gene therapy due to their efficiency and ability to stably deliver genes of interest into host cells. For example, lentiviral vectors are commonly used to reprogram somatic cells into induced pluripotent stem cells (iPS) by introducing reprogramming factors such as OCT4, SOX2, KLF4, and c-Myc.
The advantages of viral transduction over non-viral transfection make it a valuable method in biomedical research and the development of gene therapies and are summarized in Table 1 above. In contrast, the main disadvantages are also reported to provide a more comprehensive and balanced view of viral vectors compared to non-viral transfection methods.
| Advantages | Disadvantages |
| Allow more efficient gene transfer in both in vivo and in vitro environments | Can trigger severe immune responses and inflammatory reactions |
| Persist for longer periods in most cases | Their cloning capacity is limited |
| Can target a greater number of cells | Produced using complex production methods |
| A wide variety of viruses is available | Low tropism capacity for certain specific target cells |
| Innate ability for tropism towards infection | Can cause mutagenesis by inserting their exogenous DNA into the host genome |
| Capable of escaping endosomes through various evolutionary learned mechanisms | Further research is needed to better understand virus molecular infection mechanisms |
Table 1. Advantages and disadvantages of viral vectors for genetic modification.
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- Butt MH. et al. Genes (Basel) 2022 Jul 30;13(8):1370.
What types of cells can be transfected using your transfection tools?
All types of cells can be transfected using our transfection tools. We have indeed developed reagents that allow for genetic modification of both primary cells and cell lines, whether they are adherent or in suspension.
- Regarding primary cells, we have developed a proprietary technology based on a physical principle associated with chemical molecules: Magnetofection. This involves using the attractive force delivered by a specific magnetic field to attract to the cell surface complexes formed by magnetic nanoparticles and the molecules to be delivered. Briefly, once the nucleic acid/magnetic nanoparticle complexes are formed (simply by incubation for 20 minutes at room temperature), they are directly added to the cell culture medium. The cells are then placed for 20 minutes on a magnetic plate that develops a specific field compatible with Magnetofection. This causes the magnetic complexes to be attracted to the cell surface where they aggregate into clusters and cross the membrane through classical endocytosis pathways. The force is powerful enough to attract the vectors without piercing the cell membrane; thus, the cell integrity is preserved, making this method one of the most effective and least toxic to date. We offer various agents to transfect any type of primary cell with plasmid DNA: for example, NeuroMag for neurons; Glial-Mag for microglia; and PolyMag Neo for other cell types. Additionally, depending on the molecules to be transfected into the primary cells, we offer different reagents such as those mentioned above or SilenceMag to deliver small nucleic acids like siRNA. For your information, we also offer reagents for viral transduction (retro/lentivirus and adenovirus – respectively ViroMag RL and AdenoMag). The advantage of this method, which makes it more suitable for primary cells that are inherently difficult to transfect, lies in the fact that almost the entire transfected dose comes into contact with the cell surface within the first few minutes. This allows (a) to avoid an immune response from the cells that would prevent any modification of neighboring cells, and (b) to use less material for the same or even better efficiency, reducing cellular activation and toxicity.
- Regarding cell lines, we offer more "classic" transfection reagents based on lipid compounds or polymers. They are built around the same structure: a hydrophobic tail, a degradable linker, and a polar head. Thus, our products are biodegradable, which helps reduce the inherent toxicity associated with transfection agents. Similarly to primary cells, we have transfection reagents for plasmid DNA that are general-purpose, such as DreamFect Gold, specific, such as HelaFect, or intended for different nucleic acids, such as Lullaby for siRNA.
Finally, it is worth noting that we also offer transfection agents used to transfect any type of cells, whether primary or immortalized, such as the Ab- or Pro-DeliverIN reagents, for delivering antibodies and proteins, respectively, or RmesFect, designed to transfect mRNA.
* It should be noted that there is a particular type of cells that are difficult to transfect with synthetic vectors: immune cells cultured in suspension (e.g., T cells, B cells, etc.). The methods and reagents mentioned above, although described to work, do not achieve the same levels of efficiency as other cell types. Therefore, we generally recommend for these cell types to use a viral method coupled with Magnetofection or our LentiBlast Premium reagent.