Frequently Asked Questions

Can I perform DNA transfection in presence of serum?

In contrast to many other transfection reagents, the efficiency of jetPRIME® is increased when used in the presence of serum. The complexes can therefore be added directly to the cells in serum-containing medium

Can I use antibiotics during transfection?

None of our products transfection efficiency is affected by the presence of antibiotics. For example, the routine validation protocol for each new batch of jetPRIME® is always performed in serum and antibiotics containing medium.

What is the optimal cell density for DNA transfection?

Cell density will affect transfection performance. For optimal transfection with jetPRIME®, we recommend transfecting cells at 60% to 80% confluency. Please refer to Table 1 in the jetPRIME® transfection protocol for the recommended number of cells to seed according to the culture plate format. In addition, if cells have been in culture for a long time (over 20 passages), we recommend starting a new culture with a fresh tube of cells from liquid nitrogen.

How many cell passages are optimal for transfection?

Passage numbers will affect transfection performance. If cells have been in culture for a long time (over 20 passages), transfection will decrease and we recommend starting a new culture with a fresh tube of cells from liquid nitrogen. On the other hand, cells should be passaged at least 2 times after thawing before using them in transfection experiments.

How should I proceed when I want to cotransfect several plasmids in the same experiment, for example for virus production?

For cotransfection of multiple plasmids, the total DNA amount per well should not exceed the DNA amount indicated in the protocol. The ratio to use for each plasmid depends on the size of the plasmid, the plasmid construct and the expected expression level (of each plasmid). Each plasmid should represent at least 10% of the total DNA amount per well.

How to improve cell viability of fragile cells (e.g. primary fibroblasts)?

Transfection is quite a traumatic event for the cells. In order to improve cell viability, find below several suggestions:

–          change the medium 4h after transfection

–          decrease the DNA amount

–          decrease the volume of jetPRIME®

–          perform transfection in serum-containing medium

–          analyze the transfection efficiency 24 h after transfection instead of 48 h

–          ensure that jetPRIME® and DNA are diluted into the jetPRIME® buffer

–          check if the plasmid is endotoxin free

Which transfection reagent should I use for Virus production?

jetPRIME® is the reagent of choice for virus production in classical medium as detailed here. Indeed, transfection efficiency using jetPRIME® reaches up to 90% in cells commonly used for virus production, such as HEK-293 and derivatives, CHO, VERO, WOP and BHK cells, hence leading to high viral titers (Dewannieux, M., Vernochet, C., Ribet, D., Bartosch, B., Cosset, F. L., Heidmann, T. (2011). The mouse IAPE endogenous retrovirus can infect cells through any of the five GPI-anchored Ephrin A proteins., PLoS Pathog 7, e1002).

Which transfection reagent should I use for transfection of oligonucleotides (ODN)?

We usually recommend using INTERFERin® or jetPEI®, depending on the size of the oligonucleotides. jetPRIME® is not the reagent of choice for ODN transfection. Please contact the technical support if you wish to perform ODN transfection (contact us).

Which transfection reagent should I use for siRNA transfection?

INTERFERin® is the reagent of choice for siRNA transfection, and works with as little as 1 nM siRNA. jetPRIME® is aso perfectly suitable for siRNA transfection, using 10 to 50 nM siRNA final concentration.

Which transfection reagent should I use for DNA transfection into suspension cells?

Suspension cells, such as T and B lymphocytes, are notoriously hard to transfect cells with DNA, whatever the chemical reagent used. Hence we usually recommend using electroporation or virus transduction for nucleic acid delivery into suspension cells.

Which transfection reagent should I use for DNA transfection into plant cells?

Vegetal cells have a cellulose rich-membrane that prevents jetPRIME®/DNA complexes entering the cells. Even at the protoplasts stage where the cellulose envelope has been removed, complexes are not able to penetrate into the cytoplasm.

Which transfection reagent should I use for transfection into HUVEC cells?

For HUVEC cells, we recommend using jetPEI®-HUVEC, a reagent specifically designed for this purpose as described here.

Which transfection reagent should I use for transfection into primary neurons culture?

Neurons are very difficult to transfect mainly because they do not divide in culture. However, jetPEI® has been successfully used for the transfection of neurons in vitro. Our team has developed has developed a specific protocol for DNA transfection of primary neurons. Please contact our technical support for more details.

Which transfection reagent should I use for transfection into macrophages?

RAW 264.7 are successfully transfected with DNA using jetPRIME®. A transfection efficiency of 50% can be achieved following the standard conditions. For DNA transfection into primary macrophages or monocyte/dendritic cell-derived macrophages, jetPEI®-macrophage is the reagent of choice. Please click here for more information concerning a wide variety of cells.

What type of compound is jetPRIME®?

jetPRIME® is a novel proprietary cationic polymer-based molecule, synthesized at Polyplus-transfection.

How is the plasmid delivered when transfection is performed with jetPRIME®?

jetPRIME® forms positively charged complexes with DNA. These complexes then penetrate the cell through endocytosis. Endosomes then release DNA in the cytosol via the proton sponge mechanism. The plasmid mostly reaches the nucleus when the nuclear envelope disappears during mitosis.

How should jetPRIME® be stored?

jetPRIME® is a very stable molecule. jetPRIME® and its buffer are shipped at room temperature and should be stored at 4°C to ensure long term stability.

When I received my jetPRIME® vial, it was completely warm. Is it still efficient for further use?

jetPRIME® is a very stable molecule. Experiments were conducted in house to test the stability of the jetPRIME® reagent. After 48 hours at 50°C, jetPRIME® shows a similar transfection efficiency as if stored at 4°C.

I have accidentally frozen my jetPRIME® vial. What should I do?

jetPRIME® can undergo an accidental freeze/thaw cycle without compromising the transfection efficiency. However, for long term storage we recommend storing the reagent at 4°C to ensure long term stability.

How long can I use my jetPRIME® vial for?

jetPRIME® cat#114-01 (0.1 ml) is stable for 6 months. Other pack sizes of jetPRIME® are stable for at least one year, when stored appropriately.

What does the DNA/jetPRIME® ratio mean?

The ratio gives the number of microliters of jetPRIME® to use per microgram of DNA. For example, a 1:2 DNA to jetPRIME® ratio means 2 µl jetPRIME® per µg DNA.

How can I improve transfection efficiency?

The first step of optimization is to increase the DNA amount up to 1.5 fold. We also recommend increasing the DNA to jetPRIME® ratio (2 to 3 µl jetPRIME® per µg DNA). Another method consists in gentle centrifugation of the culture plate (5 min at 210 g).

Can I use the same conditions for every cell line?

The protocol provided with jetPRIME® has been optimized for a large variety of cell lines such as A549, MCF7, U-2 OS, NIH-3T3, B16-F10, Caco-2… However specific protocols have been developed for specific cell lines in order to achieve a higher efficiency. These specific conditions can be viewed in the Polyplus-transfection Database.

HEK-293 and HeLa cells: Are there any specific recommendations using jetPRIME® onto HEK-293 and HeLa cells?

jetPRIME® is very efficient for DNA transfection onto HEK-293 and HeLa cells. Hence, we recommend decreasing the DNA amount (from 1 µg to 0.5 µg per well for a 6 well plate for example), and using a 1:2 DNA to jetPRIME® ratio.

How do I scale up a transfection experiment with jetPRIME®?

In general, the amount of DNA should be proportional to the total number of cells. Refer to the regular jetPRIME® protocol indicating the amount of DNA and jetPRIME® needed according to the size of the culture dish used (Table 2).

How long are the jetPRIME®/DNA complexes stable for?

Optimal transfection efficiency is obtained with complexes formed for 15 to 30 minutes. Hence, for High Throughput Screening applications requiring longer incubation times, we recommend using jetPEI® for HTS as jetPEI®/DNA complexes are stable up to 4 hours.

Is there a plasmid size restriction for DNA transfection using jetPRIME®?

There is no size restriction for DNA transfection using jetPRIME®. However, keep in mind that for the same amount of DNA, the gene copy number will be less for large DNA than for smaller plasmids.

Do you have any specific conditions for given cell lines?

Please feel free to browse the Polyplus-transfection Database, in which specific conditions for many cells may be found.

What is INTERFERin®?

INTERFERin® is a new generation of non liposomal cationic amphiphile transfection reagent. It was developed for the delivery of siRNA into mammalian cells in culture. INTERFERin® provides more than 90% silencing efficiency at 1 nM siRNA.

Which type of cell lines has INTERFERin® already been successfully tested on?

INTERFERin® has been successfully used on various adherent and non-adherent cell lines such as HeLa, HEK-293, A549, MDA-MB-231, BHK-21, CHO and OVCAR-3. INTERFERin® has also been used on several primary cells such as primary human fibroblast and primary human hepatocytes. For more details, please refer to the Polyplus-transfection Database.

Do you recommend using INTERFERin® for miRNA transfection?

Several types of molecules are abusively called miRNA. The term miRNA can refer to:

– mimic miRNA: these are structurally similar but not identical to siRNA and can be transfected with INTERFERin®.

– plasmid encoding for miRNA: this can be transfected using jetPRIME®.

– antimiR: this is a single-stranded nucleic acid designed to specifically bind to and inhibit endogenous microRNA; it can be transfected using INTERFERin®.

Can I use INTERFERin® for in vivo applications?

INTERFERin® is dedicated only to in vitro transfections. For siRNA delivery into animals, we recommend using in vivo-jetPEI®, which has been successfully used for siRNA and oligonucleotides delivery into a wide panel of animals. Please to the in vivo-jetPEI® product page for more information

How should INTERFERin® be stored?

INTERFERin® should be stored tightly capped at 4°C.

I accidentally froze my vial of INTERFERin® at -20°C. Is it still efficient for further use?

INTERFERin® should not be frozen, as this strongly compromises its efficiency. We recommend discarding the frozen tube and using a new one.

How long can INTERFERin® be kept?

 

INTERFERin® cat#409-01 (0.1 ml) is stable for 6 months. Other pack sizes of INTERFERin® are stable for at least one year, when stored appropriately

What is PULSin® reagent?

PULSin® contains a cationic amphiphile molecule capable of forming complexes with many proteins, including antibodies or peptides, resulting in a positively charged coat. These PULSin®/proteins complexes bind to syndecans, a molecule presents in virtually all cells membrane, allowing entry in the cell and subsequent escape of the protein from the endosome to the cytoplasm.

Which cells can be used for protein delivery with PULSin®?

Established cell lines from various types such as HeLa and NIH-3T3, suspension cells such as Jurkat and primary cells such as human keratinocytes have all been successfully tested for PULSIN®-mediated  delivery of r-phycoerythrin, the positive control protein included in the kit (for an exhaustive list please refer to the Polyplus-transfection Database).

Which proteins have been delivered using PULSin®?

Negatively charged protein at physiological pH (protein with pI ranging from 0 to 7.4) with size ranging from 40 to 250 KDa have been successfully delivered using PULSin®. Moreover, various antibodies recognizing different cell components such as anti-giantin, anti-vimentin and anti-tubulin have also been successfully delivered still recognising their target. Peptides have also been successfully delivered (for details please refer to PULSin® page).

With PULSin®, is the antibody or protein only delivered to the cytoplasm?

Antibodies, proteins or peptides delivered with PULSin® are transported to the cytoplasm of the cell where it is released from the PULSin®/protein complexes. Proteins < 50kDa should be able to diffuse to the nucleus. For nuclear targeting of larger proteins, the proteins need to carry a nuclear localization signal (NLS).

I accidentally froze my vial of PULSin® at -20°C. Is it still efficient for further use?

PULSin® should not be frozen, as this strongly compromises its efficiency. We recommend discarding the frozen tube and using a new one.

What is the difference between jetPEI® and in vivo-jetPEI®?

jetPEI® (for in vitro use) and in vivo-jetPEI® are based on the same active molecule, however they have been specifically developed for each application. Thus they differ in formulation, concentration, purity and validation tests (in vivo-jetPEI® has been tested for absence of endotoxin and validated for in vivo gene delivery in mice).

How should I store in vivo-jetPEI®?

in vivo-jetPEI® is very stable and shipped at room temperature. However, for long-term conservation, we recommend storing in vivo-jetPEI® at -20°C upon reception.

What kind of nucleic acids can be delivered in vivo with in vivo-jetPEI®?

Different types of nucleic acids have been successfully delivered using in vivo-jetPEI®: DNA, siRNA, mimic miRNA, antimiR, oligonucleotides… Please refer to the Polyplus-transfection Database.

In which animals can in vivo-jetPEI® be used?

in vivo-jetPEI® has been tested in many species such as mice, rats, mosquitoes, shrimps, guinea pigs, macaques… Please feel to contact the technical support for more information (contact us).

Which administration routes can be used for in vivo-jetPEI®-mediated nucleic acid delivery?

In vivo-jetPEI® is suitable for systemic and local nucleic acid delivery. Several ways of administration have been successfully tested such as intravenous injection (tail vein and retroorbital), intraperitoneal injection, intratumoral injection, intracerebral injection, intranasal instillation, as detailed in the Application note Publications by Administration route.

I would like to target the lung. What is the best injection route?

In vivo-jetPEI® is the reagent of choice for nucleic acid delivery to the lung. Indeed, the lung is the most targeted organ following systemic delivery of in vivo-jetPEI®/nucleic acid complexes.

I want to target a specific organ; how should I proceed?

The delivery procedure highly depends on the organ to target. Depending on the route of administration, in vivo-jetPEI®-mediated gene expression was observed in the brain, liver, pancreas, spleen, kidney, heart, bladder, skin, retina, etc. Please contact our technical support team for more advice (contact us).

What is the best reagent for DNA HTS applications?

jetPEI® transfection reagent is particularly well suited for automated or manual HTS with three protocols available: reverse, batch and forward. jetPEI® provides highly reproducible results and the DNA/jetPEI® complexes are stable for up to 4 hours. Please click here for more information.

What is the best reagent for siRNA HTS applications?

INTERFERin®-HTS is a new generation siRNA transfection reagent especially developed for HTS applications providing great silencing efficiency, excellent reproducibility and high cell viability with very low amounts of reagent. INTERFERin®-HTS is cost-effective, easy to handle, compatible with serum and antibiotics, and comes with reverse and forward protocols for 96- and 384-well plates, as described here.

Can I use INTERFERin®-HTS for standard siRNA transfections?

INTERFERin®-HTS is not the reagent of choice for standard siRNA applications. Please choose INTERFERin® siRNA Tranfection Reagent for day-to-day transfections.

What is the molecular weight of jetPEI®?

jetPEI® is composed of a linear polyethylenimine (PEI) whose molecular weight is proprietary information. Extensive optimization experiments have been performed in order to select the linear PEI giving the optimal DNA delivery with the most reduced toxicity in vitro.

Can I use jetPEI® for other applications than HTS?

jetPEI® can be used for other applications than HTS such as in vitro DNA transfection. In addition Polyplus-transfection developed a specific protocol for jetPEI®-mediated DNA transfection into primary neurons. Please contact the technical support team for more information (contact us).

Can I use FectoPRO® for virus production?

FectoPRO® is perfectly well suited for virus production in suspension cells. PEIpro® is better suited for virus production in adherent cells and is provided with extensive QCs, making it suitable for therapeutic virus production.

 

What reagent should I use for bioproduction?

FectoPRO® and PEIpro® can both be used for biomanufacturing. FectoPRO® is particularly suited to achieve very high protein yields in CHO or HEK-293 growing in suspension. But for large or very-large scale experiments, you might want to use PEIpro® as it is also very efficient in HEK-293 cells and more economical.

Is the FectoPRO® booster mandatory in my bioproduction experiments?

FectoPRO® can be used without FectoPRO® booster, but it is recommended if you want to achieve high protein yields using low DNA amounts.

I have accidentally frozen my FectoPRO® vial. What should I do?

FectoPRO® can undergo an accidental freeze/thaw cycle without compromising the transfection efficiency. However, for long term storage we recommend storing the reagent at 4°C to ensure long term stability.

What is the molecular weight of PEIpro®?

PEIpro® is composed of a linear polyethylenimine (PEI) whose molecular weight is proprietary information. Extensive optimization experiments have been performed in order to select the linear PEI giving the optimal DNA delivery with the most reduced toxicity in vitro.

What is the PEI concentration of PEIpro®?

PEIpro® is provided as an aqueous solution of highly pure and qualified linear PEI at a concentration of 1 mg/L

I have accidentally frozen my PEIpro® vial. What should I do?

PEIpro® can undergo an accidental freeze/thaw cycle without compromising the transfection efficiency. However, for long term storage we recommend storing the reagent at 4°C to ensure long term stability.

What is the best solution to transfect hard to transfect cells?

Cells known as hard to transfect usually correspond to primary cells or cell lines that have slow to non dividing rates. While viral transduction constitutes a useful alternative to DNA transfection, there are several limiting factors including potential safety hazard, low titer production and higher risk of genomic integration. Therefore, we have developed an efficient non viral alternative based on messenger RNA (mRNA) delivery to outperform DNA transfection in a wide variety of cells.

What are the advantages of performing messenger RNA (mRNA) transfection?

The success of mRNA delivery is mainly due to circumventing the need for nuclear import of DNA, hindered in slow to none dividing cells. Therefore, by switching to mRNA transfection, cells that are slow dividing or that have developed specific mechanisms to protect their genome can finally be used for gene expression, with unmatched transfection efficiencies.

What are the key advantages with jetMESSENGER™ for mRNA transfection?

jetMESSENGER™’s protocol is simple: the reagent is ready-to-use and is compatible with the standard growth media (with or without serum and antibiotics) of a wide variety of adherent and suspension cell lines. jetMESSENGER™ operates through a very gentle process and upholds cell viability and overall cellular morphology in a variety of hard to transfect cells, such as rat cortex neurons, human liver carcinoma cells and mouse embryonic stem cells.

How do I obtain messenger RNA (mRNA)? Should I order it, or can I prepare my own?

mRNA can be produced using commercially available solutions (e.g. HiScribe kit provided by New England Biolabs) or be purchased “custom made” through different oligonucleotide suppliers such as Trilink, Eurofins or Aldevron.

If I choose to synthesize my own messenger RNA (mRNA), how do I start?

mRNA is synthesized from DNA templates such as plasmid DNA, PCR product, synthetic oligonucleotides. Importantly, the template must be linearized and contain a RNA polymerase promoter region such as T7 or SP6 upstream of the sequence to be transcribed, in 5’ for the sense RNA or in 3’ for the antisense RNA. If required, the RNA polymerase promoter region can be added upstream of the sequence to be transcribed by PCR using primers containing the minimal promoter sequences.

Is there a limit in messenger RNA (mRNA) size that can be obtained using mRNA synthesis kits?

There is no limit in mRNA size per se. It is worth to keep in mind that mRNA synthesis kits are usually optimized for mRNA transcripts ranging in size from 0.3 to 5kb, equivalent to a protein size of 10-150 kDa. Therefore, for shorter or longer mRNA transcripts, additional optimization may be required to obtain a similar high yield.

Are there solutions to increase messenger RNA (mRNA) stability, and is it always necessary?

During the synthesis of endogenous eukaryotic mRNA, a 7-methylguanosine CAP is added in 5’ on the nascent transcript to protect it from degradation and increase translation rate. The presence of a polyA-tail in 3’ of nascent mRNA further protects from degradation and plays a role in the nuclear export of the mature mRNA. Therefore, existing mRNA synthesis kits usually offer solutions to improve stability of mRNAs by incorporation of 5’ cap analog, and 3’ polyA-tail. In addition, specific base modification within the mRNA sequence such as 5-methylcytosine and/or N6-methyladenine can be incorporated for stability and immunogenicity purposes. Custom-made mRNA ordered from an oligonucleotide provider can be ordered with specific modifications such as Pseudo-Uridine for increased stability.

Increasing the stability of the RNA transcript may not always be necessary. For example, some viral RNA transcripts do not need to be capped and polyadenylated in order to start replicating and/or produce infectious virus after transfection.

How long will I observe messenger RNA (mRNA) expression post-transfection using jetMESSENGER™?

In comparison to DNA transfection, mRNA-mediated transfection with jetMESSENGER™ enables a more controlled gene expression that is not promoter dependent, over a similar period of time. In actively dividing cells, mRNA expression will be more prominently and stably observed between 1 to 4 days post-transfection. In slow to non-dividing cells, mRNA expression can be stable over a longer period of time, as for example up to 7 days in primary neuronal cells.

 

Can I generate stable clones by mRNA transfection alike DNA transfection?

mRNA delivery is suited for transient expression, but not for the generation of stable clones. mRNA cannot integrate into the cell genome, which prohibits the generation of stable clones but has the advantage of not modifying the genome of the host cell.

Do you have any specific conditions for mRNA transfection in given cell lines?

Please feel free to browse the Polyplus-transfection Database, in which specific conditions for many hard to transfect cells may be found.

Is messenger RNA (mRNA) transfection suited for primary cells?

mRNA transfection is perfectly suited for primary cells, including primary neuronal cell types, stem cells, immune cells and fibroblasts.

Can I perform messenger RNA (mRNA) transfection on cells grown in suspension?

jetMESSENGER mRNA transfection reagent is particularly well suited for transfection of cells grown in suspension such as Jurkat cells.

Can I perform reverse transfection with jetMESSENGER™?

jetMESSENGER™ mRNA transfection reagent can be used with two protocols: reverse and forward. In the forward protocol, the cells are split the day before transfection and the jetMESSENGER™/mRNA complexes are added to the cells. Conversely, in the reverse protocol, mRNA/jetMESSENGER complexes are first deposited in the wells prior to addition of the cells. The full protocol is available in our Resources section.

Which cells are best suited for mRNA transfection?

mRNA transfection is not only suited for difficult to transfect cells, but can also be used to transfect fast dividing and usually known as easy to transfect cells. Moreover, mRNA transfection with jetMESSENGER is suited for both adherent and suspension cell lines and primary cells.

Typically, how much mRNA is required for transfection with jetMESSENGER™ in comparison to the amounts I usually use for DNA transfection using main competitor L2K?

Lower amounts of mRNA and reagent in comparison to DNA transfection with L2K are required to perform transfection using jetMESSENGER™. Typically, per well of a 6-well plate, 2 ug of mRNA is complexed with a volume of 4 ul of jetMESSENGER™ reagent, whereas up to 2.5 ug of DNA with 5-12.5 ul of L2K are recommended.

Is it possible to get a free sample to jetMESSENGER?

A free trial pack size of 0.1 ml of jetMESSENGER™ mRNA transfection reagent is available upon request and is sufficient to perform at least 25 transfections in 6-well plates.