Posts Tagged ‘siRNA’

Sustainable RNA Transfection

 :: Posted by American Biotechnologist on 07-26-2010

MIT scientists Matthew Angel and Mehmet Fatih Yanik have discovered a method for transfecting mRNA into fibroblasts without triggering the immune response that normally defends cells against exogenous RNA infection. Cells are usually able to differentiate between endogenous and exogenous RNA through activation of pattern-recognition receptors (PRRs) that initiate a subsequent immune response. While this immune response is important for defending cells against unwanted viral RNA invasion, it also serves as a barrier for scientists interested in delivering protein-encoding mRNA into cells for a variety of purposes.

Why the need to deliver mRNA into cells? Why not just deliver DNA as is normally done utilizing traditional transfection techniques? Or, better yet, why not just skip the translational step altogether and deliver protein directly to the cells?

According to the study’s authors, since DNA is incorporated into the genome it runs the risk of causing genomic disruption which may limit its therapeutic potential while protein transduction tends to be an expensive and inefficient process. In their extensive review on the use of RNA vaccines in cancer treatment, Bringmann et al discuss how RNA has several advantages including its ability to be generated in large quantities, to be easily degraded, to not be integrated into the genome and to be quickly cleared out of the organism. As such, RNA has the potential to be a great therapeutic tool that is unencumbered by some of the pathogenic risks inherent in other molecular therapies.

Yet, obtaining sustained protein expression following mRNA transfection is challenged by the innate immune response generated by exogenous RNA. In this study, published in PLoS ONE earlier this month, Angel and Yanik show how an siRNA cocktail of interferon-β (Ifnb1), Eif2ak2, and Stat2 inhibits the RNA-mediated immune response and enables repeated mRNA transfection and subsequent protein expression.

While this particular study focused on reprogramming techniques to generate autologous pluripotent stem cells, the methodology could likely be utilized in a variety of experimental conditions focused on obtaining expression of active proteins in an inexpensive and sustainable manner.

For more information see Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins

Angel M, & Yanik MF (2010). Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins PLoS ONE, 5 (7)

Simplifying Gene Knockdown Experiments

 :: Posted by American Biotechnologist on 05-17-2010

If you’ve ever done gene knockdown experiments, you know that the process can be complicated and involve many steps. siRNA design, cell culture, perfect cell confluency, transfection or electroporation, downstream analysis etc. Bio-Rad now has a plethora of instruments and reagents to help you simplify the process. In a recently published article in the Journal of Visualized Experiments, scientists demonstrated a pathway study that makes use of Bio-Rad’s many strengths in the area of cell culture and gene silencing including streamlining cell counting with the TC10 automated cell counter, electroporating multiple samples simultaneously using the MXCell electroporation system and simultaneously assessing RNA quality and quantity with the Experion automated electrophoresis system.

If you want to learn about more ways to simplify your siRNA experiments checkout:

McCoy, A., Litterst, C., Collins, M., & Ugozzoli, L. (2010). Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells Journal of Visualized Experiments (38) DOI: 10.3791/1904

How to Perform Highly Efficient Reverse siRNA Transfection

 :: Posted by American Biotechnologist on 05-03-2010

Short interfering RNAs (siRNAs) are powerful tools to suppress gene expression in mamalian cells. While siRNA transfection can often be bogged down by cell seeding and density the highlited study demonstrates a highly efficient reverse siRNA transfection protocol for A549 cells using Bio-Rad’s siLentFect lipid reagent that does not require prior seeding of cells. Furtheromore, the simplicity of the protocol makes it suitable for high-throughput transfection. The authors acheived an average of 94% knockdown for the two genes that were targetted without impairing cell viability.

<highly efficient reverse siRNA transfection

Delivering siRNA into Difficult to Transfect Cells Using Bio-Rad’s MXCell Electroporator

 :: Posted by American Biotechnologist on 03-31-2010

Small interfereing RNA (siRNA) technology is a powerful tool for facilitating post-transcriptional gene silencing. As with most transfection techniques, siRNAs have been traditionally delivered into the cell by one of four transfection techniques: lipid-mediated, chemical-mediated, viral transfection or electroporation.

In this paper, a team from the Beckman Research Institue of the City of Hope demonstrate how Bio-Rad’s MXCell electroporator can be used to optimize transfection conditions for B-Cell lymphomas which are extremely difficlut to transfect by most traditional methods.

Delivering siRNA into Difficult to Transfect Cells Using Bio-Rad’s Gene Pulser MXcell Electroporation System