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· Why Cell Type-Specific Avalanche® Transfection Reagents?
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· Other Questions about Transfection
Other Questions about Transfection

What is transfection:

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.

Could you give me some examples of Transfection:

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.

What is the difference between stable and transient transfection:

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.

What is RNA transfection? Why people do RNA transfection?

Transfecting mRNA has several merits over DNA transfection. The merits include no risk of integration into the host genome, cell cycle-independent transfection efficiency, no need for immune inducible vectors, and adjustable and rapid expression. Using mRNA transfection, one can introduce any number of mRNAs into a cell, thereby overcoming overexpression of the genes. These advantages mostly originate from the fact that mRNA does not need to be located in a nucleus to be expressed. Transfected DNA must carry a host cell or tissue-specific promoter to be transcribed to mRNA and the expression level is determined by strength of the promoter. In contrast with DNA transfection, one can adjust expression levels by changing the amount of mRNA transfected and the frequency of transfection in mRNA transfection. Other strong advantages of mRNA transfection are:

  • 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.

What is RNAi?

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.

How long should I wait after thawing my cells before transfecting them?

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.

What is Combinatorial Chemistry, and its application on developing Avalanche™ Transfection Ragents?

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 with 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 did EZ Biosystems develop Avalanche™ Cell type/cell line specific transfection reagent series?

How soon after transfection can I assay for gene expression?

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

In what buffer and at what concentration should my DNA be suspended prior to diluting it into the Transfection Medium

Typically, our DNA is diluted in sterile water or TE buffer at a concentration of 0.5 µg/µl.

How cytotoxic are the Avalanche™ Transfection Reagents?

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

How stable are the Avalanche™ Transfection Reagents?

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

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