Transfection is a procedure that introduces foreign nucleic acids into cells to produce genetically modified cells. Transfection is a powerful analytical tool for study of gene function and regulation and protein function. The introduced genetic materials (DNAs and RNAs) exist in cells either stably or transiently depending on the nature of the genetic materials.  The main purpose of transfection is to study the function of genes or gene products, by enhancing or inhibiting specific gene expression in cells, and to produce recombinant proteins in mammalian cells.

Examples are: gene therapy delivering a gene of interest into cells to cure a disease or improve symptoms; induced pluripotent stem cell (iPS cell) generation by transfecting three or four transcription factors; small interference RNA (siRNA) knock-down procedures; and production of human tissue plasminogen activator in immortalized Chinese hamster ovary (CHO) cells for therapeutic purpose.

For stable transfection, introduced genetic materials that usually have a marker gene for selection (transgenes) are integrated into the host genome and sustain transgene expression even after host cells Replicate. In contrast with stably transfected genes, transiently transfected genes are only expressed for a limited period of time and are not integrated into the genome. Transiently transfected genetic materials can be lost by environmental factors and cell division, so the choice of stable or transient transfection depends on the objective of the experiment.

• Transfected mRNAs can be expressed within minutes after transfection because it skips translocation to the nucleus and transcription process; and
• Transcriptome (population of mRNAs) can be used for transfection, which is practically difficult in DNA transfection.

For these reasons, transfecting RNA is attracting interest for therapeutic purposes.

RNA interference (RNAi) is a powerful tool to knockdown specific genes and to observe consequent changes of phenotypes. Introduced small inhibitory RNAs (siRNA) form RNA-induced silencing complex (RISC) in the cell and the RISC inhibits the expression of target gene expression. The most common methods used to deliver siRNA are lipid/polymer-mediated delivery and virus mediated delivery. Despite the wide use of siRNA, large efforts are still being made to develop more effective, safe, and reliable methods to deliver siRNAs into cells, because of the great potential of RNAi in clinical use to treat diseases . Both relatively new transfection methods, mRNA and siRNA transfection, lead to new ways to execute cell research with their own distinctive advantages.

We strongly recommend transfecting cells after at least one passage but no longer than three passages. Ideally, your cells should be transfected when they are at the peak of health, which is typically 70-80% confluency.

Combinatorial chemistry involves the rapid synthesis or the computer simulation of a large number of different but often structurally related molecules or materials. In a combinatorial synthesis, the number of compounds made increases exponentially with the number of chemical steps. In a binary light-directed synthesis, 2n compounds can be made in n chemical steps. Combinatorial chemistry is especially common in CADD (Computer aided drug design) and can be done online with web based software, such as Molinspiration. Here in EZ Biosystems, We used Combinatorial Chemistry, together with other technologies, to develop thousands of new candidate ingredients that were later used to screen for effective ingredients by using our High Throughput Screening technology for our Cell Type/Cell Line Specific Avalanche® Transfection Reagents series. For details about the developing process, go to:  How Were Cell Type-Specific Avalanche® Transfection Reagents Developed?

We assay for gene expression 24 or 48 hours post-transfection for both primary cells and cell lines.

Typically, our DNA is diluted in sterile water or TE buffer at a concentration of 1.0 µg/µl.

The Avalanche® Transfection Kits have very low cytotoxicity compared to leading competitor’s transfection reagents and compared to electroporation.

All Avalanche® Transfection Reagents are stable for 1 year when stored properly.

Avalanche®-Omni Transfection Reagent is an exceptionally powerful and versatile next-generation DNA and siRNA transfection reagent for day-to-day experiments. It has been shown to effectively transfect the broadest spectrum of adherent and suspension cells. No other transfection reagents can match the efficiency, spectrum, convenience, and gentleness of Avalanche®-Omni Transfection Reagent for transfection of primary, challenging, and sensitive cell lines. It includes a streamlined, simple protocol where the complexes are added directly to cells without changing media. This lends itself to high throughput applications. It works very well in the presence or absence of serum.

DNA needs to be pure enough to be exposed to the cells otherwise they will lyse, or the transfection efficiency will be so low as to be ineffective. Both CsCl purification and column-based purification methods should produce high quality DNA. After any purification, be sure to remove all the ethanol before resuspending the DNA in sterile water or TE. It is highly recommended to do a “cells + DNA only” control to check for any adverse side effects of the DNA on the cells. Additionally, leave one plate with just cells and media as a control.

Optimization of transfection conditions is essential for the highest-efficiency transfections and the lowest toxicity. The conditions that should be optimized include Avalanche®-Omni Transfection Reagent and DNA concentrations, the ratio of them, and cell number etc. To optimize the amount of Avalanche®-Omni Transfection Reagent, start with cells at 80-90% confluency and 0.5 µg DNA for 24-well plates. With cell number and DNA concentration held constant, vary the amount of Avalanche-Omni® to determine the optimal concentration. The product protocol gives a simple procedure for optimization. The cell number and amount of DNA can also be optimized. It is possible to minimize the effect of transfection on cell growth and viability by increasing the number of cells plated per well or by decreasing either Avalanche®-Omni Transfection Reagent or DNA concentrations. With careful optimization this can be achieved with little impact on the level of transgene expression.

We discourage using any antibiotics during transfection (e.g. Geneticin®, Hygromycin, Gentamycin, Penicillin, etc). There can be higher cell death when antibiotics are present during transfection. Even though some such as penicillin and streptomycin are not toxic to eukaryotic cells in a healthy culture, during transfection the cell permeability increases so that much higher levels of antibiotics get into cells. For stable transfections, wait at least 24-48 h after transfection before adding selective antibiotics.

It depends on your transfection goals. If you want to have a broad spectrum transfection reagent that provides excellent transfection efficiency on most of your cell types for day-to-day experiments with minimum optimization, Avalanche®-Omni Transfection Reagent (EZT-OMNI-1) is the one that you need. If you want to achieve the best transfection result for a specific type of cells, use cell type-specific Avalanche® transfection reagents. Your first step is to go to our product list, and find out the cell type/cell line specific Avalanche® transfection reagents for your cells by identifying the respective cell types or cell lines. Use the Filters on the left to narrow down the product list. The cell type/cell line specific Avalanche® transfection reagents were specifically designed for transfection on the respective cell types or cell lines. The proprietary formulations ensure the highest possible transfection efficiencies and viabilities without comprehensive optimization. Click HERE to find out how EZ Biosystems developed the cell type/cell line specific Avalanche® transfection reagents.

In general, once optimal transfection conditions are determined for a given cell line, it is recommended that cells be passaged less than 20 times to maintain reproducible results. Thus immediately following the determination of optimal conditions, cells should be frozen down so that when the working stock approaches 20 passages, a new batch can be started from the frozen stock.

Opti-MEM® Reduced-Serum Medium (Cat# 31985-070, Life Technologies) or regular DMEM without serum.

Not in all cases. What is essential is to form the reagent:nucleic acid complex in the absence of serum, because negatively charged proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serum containing medium.

Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Supercoiled plasmid runs faster than linear plasmid. Nicked plasmid will run slower than linear plasmid.
The content of nicked DNA in your DNA preparation should be below 20%. Higher content of nicked DNA results in significant decrease of transfection efficiency.

All products of the Avalanche® Transfection Reagent series are stable at 4º C for at least one year. Do not freeze.

It is absolutely critical to have a control oligonucleotide to be able to determine any non-specific effects. This oligonucleotide can be a scrambled oligonucleotide (same length and base composition in a random order) or a sense oligonucleotide if the target is mRNA.

Yes. To check the quality of your DNA, we strongly recommend determining the A260:A280 ratio. It should be at least 1.7 for a good DNA preparation.

Yes, the general procedure below is recommended to cotransfect your plasmid DNA and an RNAi molecule into mammalian cells using Avalanche® Transfection Reagents. Detailed cotransfection methods are included in the product protocols.

One day before transfection, plate cells in the appropriate amount of growth medium without antibiotics such that they will be 80-90% confluent at the time of transfection.
For each transfection sample, prepare DNA-RNAi molecule-Avalanche® Transfection Reagents complexes as follows:
Dilute the DNA and RNAi molecule in the appropriate amount of Opti-MEM® I Medium without serum. Mix gently.
Mix Avalanche® Transfection Reagents gently before use. Add respective amount of Avalanche® Transfection Reagent into the diluted DNA and RNAi molecule . Immediately mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur.
Add the DNA-RNAi molecule-Avalanche® Transfection Reagents complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
Incubate the cells at 37 °C in a CO2 incubator until you are ready to harvest cells and assay for your target gene. Removal of complexes or media change is not required; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.