RNA interference (RNAi) is a cellular process where specific RNA molecules prevent a gene from producing its corresponding protein. Scientists have adapted this mechanism as a research tool, allowing them to temporarily “silence” the activity of a chosen gene to investigate its function. By observing the effects of reducing a gene’s expression, often by 90% or more, researchers can deduce its role within a biological system.
Designing the RNAi Experiment
The design process begins with target selection, where a researcher identifies the specific gene to be silenced. Within that gene’s messenger RNA (mRNA) sequence, a unique segment of 21 to 23 nucleotides is chosen as the target. Selecting a sequence unique to that gene is necessary to avoid unintentionally affecting other genes.
Next, the researcher chooses the type of RNAi molecule. The two primary options are small interfering RNA (siRNA) and short hairpin RNA (shRNA). siRNAs are synthetic molecules that provide a temporary reduction in gene expression, with effects lasting for several days, making them suitable for short-term studies.
For long-term gene knockdown, researchers use shRNA. These sequences are processed into siRNA inside the cell and can be integrated into the cell’s genetic material, allowing for continuous silencing. To aid in the design of both molecule types, bioinformatics software is used to identify effective target sequences and minimize potential “off-target” effects, where the RNAi molecule might accidentally silence unintended genes.
Delivery Methods for RNAi Triggers
After designing the RNAi molecule, it must be delivered into the target cells. The choice of delivery method depends on the RNAi molecule, cell type, and desired duration of silencing. For temporary knockdown using siRNAs, a common method is transfection. One popular transfection technique is lipofection, where siRNA molecules are enclosed within small, fatty vesicles called liposomes. These liposomes then fuse with the cell membrane, releasing the siRNA into the cell’s interior.
Another transfection method is electroporation. This technique applies a brief electrical pulse to the cells, which creates temporary pores in their membranes. These pores allow the siRNAs from the surrounding solution to enter the cell before the membrane reseals. Electroporation can be effective for a wide variety of cell types, including those that are difficult to transfect using chemical methods.
For long-term gene silencing with shRNAs, the preferred delivery method is transduction, which utilizes modified viruses as vectors. Viruses such as lentiviruses are engineered to be safe by removing their ability to cause disease, while retaining their natural ability to infect cells and deliver genetic material. These viral vectors carry the shRNA sequence into target cells, where it can be integrated into the host cell’s genome. This integration ensures stable and long-lasting gene knockdown.
Core Protocol Execution and Experimental Controls
The core laboratory protocol begins with plating the target cells in culture dishes and allowing them to grow to an appropriate density. The RNAi molecules are then prepared by mixing them with the chosen delivery reagent, such as lipofection chemicals or viral particles. This mixture is then added to the cells.
After introducing the RNAi trigger, the cells are incubated for a period that ranges from 24 to 72 hours. During this time, the cell’s internal machinery recognizes the delivered molecules and begins to degrade the target mRNA, leading to gene silencing. The use of proper experimental controls, run in parallel, is required to interpret the results reliably.
Several types of controls are necessary. A negative control uses a non-targeting RNAi molecule with a sequence that does not match any gene in the cell’s genome. This helps ensure that the delivery process itself or the presence of foreign RNA is not causing the observed cellular changes. A positive control uses an RNAi molecule known to effectively silence a gene, confirming that the delivery method and reagents are working correctly. An untreated control group of cells receives no treatment at all, providing a baseline for normal gene expression.
Validating Gene Knockdown
The final step is to validate that gene silencing was successful by measuring the levels of the target gene’s products. The first level of validation is measuring the amount of messenger RNA (mRNA) using Quantitative Real-Time Polymerase Chain Reaction (qPCR). This method allows for the precise quantification of specific mRNA sequences. A successful knockdown is indicated by a substantial decrease in the target mRNA in the treated cells compared to the control cells.
While reducing mRNA is the direct action of RNAi, the ultimate goal is to decrease the amount of protein. Therefore, it is also important to measure protein levels. The most common method for this is the Western blot, which separates proteins by size and uses specific antibodies to detect and quantify the target protein. A successful experiment will show a much fainter protein band for the target protein in the silenced cells compared to the control cells, confirming a functional knockdown.