Transfection Basics

The Basics of Transfection

Transfection is a process by which material is transported into a cell. Transduction is a type of transfection that viruses use to replicate themselves. When it comes to transfecting genetic materials for medicinal use or scientific research, there are many drawbacks with using viruses such as high production cost, time concerns, and immunogenicity. In an effort to alleviate these drawbacks scientists invented non-viral methods of transduction, or Transfection.

Of the many methods used for transfection, “chemical transfection” is the simplest and most widely used method. In general, this method of transfection uses specific blends of synthetically derived polymers and/or lipids that are dissolved in specialized liquids to form what are termed transfection reagents. These reagents are added to different types of genetic material that transport them across the outer membrane and to into the cell’s cytoplasm and in many cases into the nucleus. With recent “best-in-class reagents”, this process can now be performed using multiple cell types in a safe and effective manner. The mechanism-of-action (MOA) of how any given reagent transfects any given cell type is a dynamic process and beyond the scope of this basic review. What is clear is that best-in-class reagents are powerfully effective, robust, less toxic and display minimal off-target side effects.

Anyone new to this field should carefully read this entire page first and for more details the literature. To simplify your literature search, we also developed a citation database found on this website so you can find peer reviewed manuscripts specific to your cell line and application. You can also learn about FuGENE’s “best-in-class” reagent offerings on the website or by contacting FuGENE directly.

Transfection basics to keep in mind when running your transfection experiments:

Cell Types used – any rapidly dividing cell that is adherent can be transfected and the percent cells transfected should range from 40 to 100%. If you do not obtain these levels, please contact FuGENE for help. Suspension or non-dividing cell types are usually more difficult and require more advanced blends. Anyone trying to transfect these cell types should first contact FuGENE for help and information regarding the latest specialized blends.
Testing and Optimizing Transfection Efficiency – using a green fluorescent protein (GFP) gene, transfection is typically analyzed using Flow cytometry (FC) and data is shown in four sets; percent cells transfected, level of protein expressed, cell toxicity using propidium iodide (PI), and counting total the number of cells post transfection. Best-in-class reagents typically produce a high % of cells expressing GFP, a high total level of GFP, a low % of cells incorporating PI, and the number of cells post transfection being close to the number of cells observed in a none transfected control.
Reagent to Nucleic Acid (NA) Ratios – measured in microliters reagent to micrograms of genetic material with the industry standard ratio being 3 to 1 (or 3:1).
Complexation of reagent to NA – typically done in cell media without proteins or PBS (pH 7.4) and takes 15-30 minutes.
Transfection time – after complexation, reagent/NA complexes will be added to cells and transfection analyzed six hours to seven days while keeping the cells well fed and at the proper densities.
Increasing success – 1. Use freshly thawed cells, as high passage numbers can reduce protein expression, 2, optimize DNA to reagent ratios, 3. use smaller plasmids, as they transfect better, 4. synchronize cells in G2/M phase, as this will increase the percentage of cells transfected.

How to Run a Simple Transfection

Benefits of current “best-in-class” Transfection Reagents

Delivering DNA using Transfection Reagents

DNA Delivery to the Nucleus

To make RNA and proteins, transfected DNA needs to be transported into the nucleus. Thus, in addition to being transported across the plasma membrane, DNA also needs to be transported to the nuclease and cross the nuclear membrane. Intercellular transport is believed to occur via endosomes and the closer to the nucleus that endosomes release their DNA cargo, the better chance that the DNA will enter the nucleus. Reasoning for this is based on the viscous nature of the cytoplasmic material and the less chances for DNA degradation. Distance is also thought to explain why adherent cells typically transfect better than suspension cells since the distance between the outer membrane and the nuclease is shorter in adherent cells. To protect the DNA and help it cross through nuclear pores, best-in-class reagents will use polymers that complex with and compact the DNA.

Main types of DNAs used in Transfection and their Benefits

Plasmids are typically circular double-stranded DNA that are produced in bacteria. When using plasmid for transfection, plasmid size is important as smaller plasmids (6 KB) tend to lead to higher protein production than larger plasmids (15KB) (see figure below). To isolate and purify the plasmids for transfection, bacterial cells are first lysed and the plasmids isolated and cleaned using a variety of methods. To obtain best results in transfection, plasmids should be clean of cellular debris and special care taken to remove DNases and endotoxins. Today, silica-based column purification kits (Prep Kits) are commonly used and can be purchased from multiple companies.

Smaller Plasmids Yield More Protein

PCR products are typically linear double-stranded fragments of DNA made using the polymerase chain reaction (PCR) and can be used to express proteins or RNA in cells via transfection. PCR products also tend to be easier to make then plasmids since they don’t require time-consuming cloning nor bacteria. This especially is true if someone wants to test multiple variants before choosing the best one for plasmid production. One should take caution to use a high-fidelity polymerase as standard Taq polymerases can produce multiple mutations. This is especially true if multiple rounds of PCR are needed. That said, when the correct transfection reagent is used, no significant differences in the expression efficiency between PCR products and plasmids have been observed.

https://doi.org/10.1371/journal.pone.0232045

Delivering RNA using Transfection Reagents

RNA Delivery to the Cytoplasm

Huge breakthroughs in RNA transfection have taken place since the 1980s when scientists began demonstrating that transfecting RNA was a useful technology. RNA transfection gained world-wide recognition when it was shown it could be used to make coronavirus vaccines. Now researchers can use RNA transfection to regulate protein translation, express new proteins, and more recently perform CRISPR experiments without any DNA or protein.

RNA transfection is generally more gentle on cells then plasmids are, as RNA is less likely to interfere with or cause irreversible changes in the cell’s genome. In addition, with the advent of base modifications and new reagents like FuGENE RX, RNA transfection can deliver higher levels of protein in a more gentle manner then DNA can. When designing your RNA for your experiments, if high expression and/or low adverse immune responses are desired, we highly suggests to our customers to use FuGENE RX and N1-methylpseudouridine modified RNA.

Therefore, with all the recent advances in RNA transfection, anyone needing to transfect cells should definitely consider using RNA.

Main types of RNAs used in Transfection

Messenger RNA (mRNA) Transfection into cells is typically performed to express proteins. For this goal, transfecting mRNA has advantages when compared to transfecting plasmid DNA.

mRNA transfection can surpass DNA plasmids for protein expression levels when using N1MePsU modified uridines.

Advancements in RNA chemistries (modified bases and capping) and best-in-class mRNA specific reagents allows for high production of most any protein, in any cell type

mRNA overcomes undesired genome integration effects

mRNA allows for faster protein production since transport into the nucleus and transcription are not required

RNA transfection is usually more efficient than DNA at producing proteins in non-dividing cells

Advanced mRNA chemistries yield longer transcripts that do not induce adverse effects (minimal to no toxicity) and faster and long-lasting protein production (minimal degradation)

Small Interfering RNA (siRNA) is used to inhibit (knock-down) protein production. siRNA transfection is unique as it also allows the scientist to dial down the production of a single protein by simply adjusting the siRNA levels used in the transfection. Here are additional advantages of siRNA over small molecule inhibitors or CRISPR.

siRNA transfections are much more specific than small molecule inhibitors, which are plagued with off target side effects

siRNA allows for a way to modulate target expression allowing for a better understanding of function

unlike CRISPR, which can only knock-out protein expression at the DNA level, siRNA can target essential proteins that would lead to cell death using CRISPR

siRNA do not interfere with the genome eliminating undesired genome changes

Circular RNA (cRNA) has garnered attention recently as a replacement for mRNA because it appears to be safer, do not require the modifications mRNA require, it is not prone to degradation and also does not require capping. cRNA is typically made via back-splicing that can be induced by ribozyme incorporated sequences, and by definition, does not have 5’ and 3’ ends. Since cRNA is endless, to be used as a transcript, it must have internal ribosome entry sites (IRES) which are structured RNA sequences that ribosomes can recognize under certain conditions. Though IRES recognition is less efficient than 5’-cap recognition, during times of stress when ribosome proteins cease recognizing 5’-caps, IRES becomes the preferred site for entry. All these factors bring with them advantages and disadvantages as described below:

Circularization of RNA can be complicated but multiple methods are published
For the most part, cRNA is quite stable and do not require special modifications
IRES sequences are used for ribosome recognition, replacing the need for 5’-caps
IRES recognition by ribosomes mainly occurs during cell stress
To make cRNA, standard processes and T7 RNA polymerase is used
To express high amounts of proteins, cRNA will not be your best choice

Major Applications and Fields that routinely use Chemical Transfection

Transfection allows someone to increase or decrease virtually any protein within any living cell. So, it is not surprising that chemical transfection plays a large part in multiple fields including biological research, manufacturing and therapeutic development. The list and figure below show some of the main application areas that are powered by transfection.

Applications for Chemical Transfection Reagents

Gene Therapy

Vaccines

Cell Therapy

Virus Manufacturing

Protein Manufacturing

Stem Cell Manufacturing

Cell Analysis

Live Cell Imaging
Drug Target Identification / Validation

Research Fields that Use Chemical Transfection Reagents

Stem Cells

Oncology

Neurobiology

Hematology

Drug Development

Drug Research

Reference: doi:10.1016/j.ymeth.2017.10.008

It has been known for some time that viruses can infect living cells by delivering their genetic material into the cell. This process is known as transduction. When it comes to delivering genetic materials for medicinal use or scientific research, there are many drawbacks with using viruses such as high production cost, time concerns, and immunogenicity. In an effort to alleviate these drawbacks, scientists invented non-viral methods of transduction, or Transfection.

Of the many methods used for transfection, “chemical transfection” is the simplest and most widely used method. In general, this method of transfection uses specific blends of synthetically derived polymers and/or lipids that are dissolved in specialized liquids to form what are termed transfection reagents. These reagents are added to DNA or RNA and transport them to either the cell’s nucleus or cytoplasm. With recent “best-in-class reagents”, this process can now be performed in multiple cell types in a safe and effective manner. The mechanisms-of-action (MOA) of how any given reagent transfects any given cell type is a dynamic process and beyond the scope of this basic review. What is clear though is that some reagents are more effective, more robust and less toxic than others.
Anyone new to this field should to carefully read the literature, use our database to find peer reviewed manuscripts specific to your cell line or application and learn more about FuGENE’s most recent “best-in-class” reagent offerings by contacting FuGENE directly.

Transfection basics to keep in mind when learning about chemical transfection or picking the correct reagent:

Cell Types used – typically any adherent dividing cell type should transfect well, with percent of cells be transfected in a given reaction range from 40% to 100%. If you do not obtain these levels, please contact FuGENE. Suspension or non-dividing cell types are usually more difficult and require more advanced blends. Anyone trying to transfect these cell types should first contact FuGENE for help and information regarding the latest specialized blends.
Testing Transfection Efficiency – using a green fluorescent protein (GFP) gene, transfection is typically analyzed using Flow cytometry (FC) and data is shown in three sets; percent cells transfected, level of protein expressed, and cell toxicity using propidium iodide (PI), and counting total the number of cells post transfection. Best-in-class reagents typically produce a high % of cells expressing GFP, a high total level of GFP, a low % of cells incorporating PI, and the number of cells post transfection being close to the number of cells observed in a none transfected control.
Reagent to NA ratios – measured in microliters reagent to nanograms of genetic material the industry standard ratio is usually around 3 to 1 (or 3:1).
Complexation – typically is done in cell media without proteins or PBS (pH 7.4) and takes a few minutes. Typically, genetic material is clean from proteins and diluted in pure water.
Transfection time – after complexation, complexes will be added to cells and transfection analyzed in 6 – 96 hours.
Increasing success – using freshly thawed cells, using correct DNA to NA ratios, using smaller plasmids and/or synchronizing your cells can increase transfection efficiency dramatically.
Basic Steps – watch the 2-minute video showing how to perform a transfection experiment.

Delivering DNA and RNA using Transfection Reagents

Put a short into blurb here

DNA Delivery to the Nucleus

RNA Delivery to the Cytoplasm

Main types of RNAs Scientist want Transfected

RNA Delivery to the Cytoplasm

A main reason to transfect mRNA into cells is to express protein. For this goal, transfecting mRNA has advantages when compared to transfecting plasmid DNA.

Today, mRNA transfection surpasses DNA plasmids in protein expression

Advancements in RNA chemistries and best-in-class mRNA specific reagents, allows for high production of most any protein, in any cell type

mRNA overcomes undesired genome integration effects

mRNA allows for faster protein production since transport into the nucleus and transcription are not required

RNA transfection is more efficient in non-dividing cells

Recent advances in mRNA chemistries (modified bases and capping) yield longer transcripts that do not induce adverse effects (minimal to no toxicity) and faster and long-lasting protein production (minimal degradation)

Small Interfering RNA (siRNA)

The main reason to transfect siRNA into cells is to inhibit (knock-down) a protein’s production. For this goal, transfecting siRNA has advantages when compared to small molecule inhibitors or CRISPR.

siRNA transfections are much more specific than small molecule inhibitors, which are plagued with off target side effects

siRNA allows for a way to modulate target expression allowing for a better understanding of function

unlike CRISPR, which can only knock-out protein expression at the DNA level, siRNA can target essential proteins that would lead to cell death using CRISPR

siRNAs do not interfere with the genome eliminating undesired genome changes

Circular RNA (cRNA)

Though somewhat new to the field, cRNAs have garnered attention recently as a replacement for mRNAs, as they appear to be very safe, do not require the modifications that mRNA do since they are not prone to degradation and also do not require capping. cRNAs are typically made via back-splicing that can be induced by ribozyme incorporated sequences, but by definition, they must have their 5’ and 3’ ends are covalently attached. Since these RNAs are endless, to be used as transcripts, cRNAs use internal ribosome entry sites (IRES) which are structured RNA sequences that ribosomes can recognize under certain conditions. Though IRES recognition is less efficient than 5’-cap recognition, during times of stress when ribosome proteins cease recognizing 5’-caps, IRES becomes the preferred site for entry. All these factors bring with them advantages and disadvantages.

1. circularization can be tricky and inefficient but multiple methods are being explored
2. for the most part, cRNAs are quite stable but certain design constraints exist
3. 5’-caps cannot be added to cRNAs and therefore an IRES is required for translation
4. IRES recognition mainly occurs during stress conditions

cRNAs can be made naturally using standard bases and T7 RNA polymerase

to express proteins in cells, in many cases, IRES recognition may not be efficient enough

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Major Fields that routinely use Chemical Transfection

Transfection allows someone to increase or decrease virtually any protein within a cell. So, it is not surprising that transfection plays a large part in multiple fields including biological research, manufacturing and therapeutic development. The figure below shows displays the main application areas that are powered by transfection.