Optimizing the Conditions for Gene-Silencing Experiments

In the body, a single mutant gene is enough to trigger many forms of cancer or activate inherited conditions such as Huntington's disease. In the laboratory, the ability to switch a gene off or on is the key to deciphering its function and then developing ways to treat or even prevent the associated disease.

Switching genes off and on is simple in concept but difficult in practice. It started getting easier in 1998 when researchers discovered a natural mechanism called RNA interference (RNAi). Then, in 2001, follow-on work led to a technique called small interfering RNA (siRNA), which uses very short RNA segments to affect a specific portion of the genetic sequence—and can silence a specific gene in just a few days.

The speed and specificity of siRNA has transformed the world of biotechnology, impacting basic disease research, target validation and drug discovery. Some companies are even developing RNAi-based therapeutics. To further accelerate the pace of discovery, researchers can benefit from easy, effective analytical solutions that help optimize their gene-silencing experiments.

Finding the best sequence

One of the biggest challenges in RNAi is the delivery or transfection of siRNA into target cells. Successful transfection depends on a few key factors: the target cell line; siRNA concentration; the ratio of siRNA and transfection reagent; cell confluence during transfection; and incubation time and media. To further enhance the effectiveness of gene-silencing experiments, researchers need to verify the quality of the siRNA, optimize the conditions for transfection and measure the success of the transfection.

Finding the most effective snippet of RNA for each gene of interest usually requires testing of three or four different siRNA sequences. Before testing and comparing any of the sequences, researchers need to monitor and optimize three key parameters: siRNA purity and integrity; siRNA uptake; and cell viability. Once optimal transfection conditions have been established, the candidate siRNA sequences can be evaluated for their ability to decrease expression of the target protein.

Accelerating the analysis process

The Agilent 2100 bioanalyzer can automate and accelerate these tasks. With a few complementary products, the bioanalyzer becomes a powerful analysis solution for gene-silencing experiments. For example, adding the Agilent Cell Fluorescence and DNA 1000 LabChip kits enables fast, automated cell assays. Transfection agents and fluorescently tagged siRNA from companies such as QIAGEN N.V. provide the essential media and materials.

The Agilent 2100 bioanalyzer helps automate and accelerate the optimization of transfection conditions

Typical methods and results are described in the Agilent application note, "siRNA Transfection Optimization with the Agilent 2100 Bioanalyzer." The note outlines the use of the bioanalyzer to quickly verify siRNA integrity and determine the optimal transfection conditions for gene silencing—with the help of red-fluorescently labeled siRNA and cell fluorescence assays. Simultaneous evaluation of cell viability and delivery efficacy was used to determine the optimal transfection conditions.

Achieving greater efficiency and viability

Verifying the integrity and purity of the siRNA was done with the DNA 1000 LabChip kit, which needed just 25 ng of siRNA. Optimization of transfection viability required evaluation and comparison of several transfection reagents and methods. Monitoring of the transfection efficiency of fluorescently tagged siRNA was performed with the Cell Fluorescence LabChip kit.

The results were impressive. Optimizing transfection conditions for siRNA uptake and viability required just two chip runs utilizing one set of 12-well plates—all within one hour. After identifying the optimized siRNA amount and transfection agent ratio, the experiment achieved a transfection efficiency of 85% and kept more than 95% of the cells viable.

This type of fast, automated analysis accelerates exploration of the exciting insights made possible by siRNA. Whether researchers are focused on functional analysis, target validation or gene-specific therapeutics, Agilent's single-platform solution can enhance their work.

For more information

Other sections of the Agilent Web site can help you learn more about our solutions for disease discovery, drug discovery and gene silencing. For additional information about these and other Agilent life sciences products and resources, please visit the main page of the Life Sciences/Chemical Analysis section of our Web site.