RNA Interference (RNAi)

RNA interference (RNAi) is a highly conserved biological process in which RNA molecules target mRNA transcripts to cause specific gene silencing. This pathway is a crucial part of cellular defense and is also involved in regulating various physiological processes. The ability of researchers to induce specific gene silencing has revolutionized the field of functional biology. There are two main types of small RNA molecules that function in the RNAi gene silencing pathway: short interfering RNA (siRNA) and microRNA (miRNA). 

At Thermo Fisher Scientific, we have developed our Invitrogen RNAi portfolio to support in vitro through in vivo research. Our products also scale from single gene studies to libraries for high-throughput screening applications and mid-scale production. Your choice of RNAi tools depends on experimental parameters including your model system, the length of time you require knockdown, and scale. Our comprehensive portfolio of RNAi technology is designed to meet this range of needs, helping provide you with the most effective solutions for precise gene silencing.

RNAi technology takes advantage of endogenous cellular machinery, to effectively knock down expression of a gene of interest. Gene silencing via RNAi can be facilitated through the following technologies (Figure 1):

1. Synthetic RNA molecules 
2. RNAi vectors 
3. In vitro dicing 

In mammalian cells, short pieces of dsRNA, such as siRNA, initiate the specific degradation of a targeted cellular mRNA. In this process the antisense strand of the siRNA duplex is loaded into the multi-protein RNA-induced silencing complex (RISC), which then identifies the corresponding mRNA and cleaves it at a specific site. This cleaved message is targeted for degradation, which ultimately results in the loss of protein expression. 

We have developed a broad suite of tools for RNAi applications, including short interfering RNA (siRNA) for targeted gene knockdown and microRNA (miRNA) reagents to mimic or inhibit endogenous gene regulators. Discover our available solutions to help advance your RNAi research.

In addition to the proper use of controls, these guidelines outline experimental best practices for RNAi gene silencing.

Minimize off-target activity by employing at least two unique siRNA sequences targeting different regions of the same mRNA. This confirms that the observed phenotype is due to the intended gene knockdown.

Use the lowest effective siRNA concentration to achieve robust knockdown while minimizing off-target effects. Silencer Select and Stealth RNAi siRNAs are effective at low doses, reducing the impact on normal cellular processes.

Untransfected cells serve as important controls to account for media changes, assay conditions, and other variables, offering a baseline for assessing cell viability and helping ensure reproducibility.

Make sure the phenotypic changes from RNAi-mediated gene silencing are linked to decreased mRNA levels using qRT-PCR. If siRNAs induce a phenotype without reducing mRNA, it may indicate non-specific responses or off-target effects.

Validate reagents for qRT-PCR and western blots before transfection to help ensure sensitivity in detecting changes in mRNA and protein levels. This is essential for interpreting knockdown results, especially for genes or proteins with low expression levels.