RNA interference - shRNA and siRNA

RNA interference (RNAi) is a process where RNA molecules can specifically suppress the expression of proteins, called gene knockdown. Several forms of RNAi have been observed in nature, and in the last two decades RNAi has been used in biotechnology for a wide range of applications. This application utilizes cells' own processes for silencing gene expression to suppress specific targets. This technique can be used to reversibly assess the effects of gene knockdown on a cellular scale. Many gene knockdown mechanisms cannot be reversed, and in an experiment this means that it is not possible to separate outcomes caused by the knockdown from off-target effects.
RNAi can be used to silence, or 'switch off' a target gene. Allowing the RNAi to degrade causes the gene to be 'switched on' again. In an experiment, this approach can be used to demonstrate that measured outcomes (for example, changes in cell motility, protein expression etc.) are truly associated with silencing of the target gene. Paired with suitable controls, RNAi allows for near-causal manipulation of target protein expression in a cell population.

Several types of RNAi are used in biotechnology; we focus on siRNA and shRNA plasmids. 

  • Short interfering RNA (siRNAs) are duplex molecules, typically 20-24 base pairs long that are capable of inducing gene silencing.
  • Short hairpin RNA (shRNAs) are double-stranded RNA molecules that include a single hairpin turn. shRNA plasmids are introduced into the host cell and produce shRNA molecules which are processed into siRNA molecules. 

Both siRNA and shRNA forms of interference work by the production of single-stranded RNA, which forms an RNA-induced Silencing Complex (RISC) with several other proteins and enzymes. The single-stranded RNA within the complex binds to the target mRNA, slicing the mRNA. The cut mRNA is recognized as abnormal by the cell and is degraded, leading to the silencing of that gene.

siRNA duplex shRNA Plasmids

siRNA molecules do not need to be processed by the cell dicer enzymes, and are immediately available for RNAi. The target gene can be suppressed faster with siRNA than shRNA.

shRNA Plasmids usually contain an antibiotic resistance gene, which allows for antibiotic selection of cells expressing the shRNA.

Transfection is transient (RNAi occurs temporarily)

Transfection can be transient (RNAi occurs temporarily) or stable (RNAi occurs continually)

No selection step occurs with siRNA duplex transfection, meaning some cells in the population may not have been successfully transfected. Additional selection steps may be included as required.

Transfection using shRNA is a longer process. Depending on the method used, the total protocol time is 3-8 days.

RNA interference (RNAi) is a process where RNA molecules can specifically suppress the expression of proteins.

Check our available RNAi products here.

Sandwich ELISA kit information

Intracellular delivery

For RNAi to occur, the interfering RNA molecules must enter the target cell. Several techniques have been developed to deliver the RNAi molecules into cells:

  • Transfection: This method directly introduces the RNAi molecules into the host cell, which can be achieved through several approaches:
    • Lipofection: This method uses liposomes, small vesicles comprised of a phospholipid bilayer. The RNAi molecules are packaged into the liposomes. When liposomes are introduced to the host cell, they merge with the cell membrane and release the RNAi molecules to the cell. This is not a particularly toxic method.
    • Viral Transfection: RNAi molecules are packaged into viral vectors (typically lentivirus and adenovirus), which deliver the RNAi molecules directly to the cells. Viral delivery is a useful method when cells are resistant to other forms of transfection, but can sometimes lead to immune responses that interfere with results.
  • Electroporation: Target cells are mixed with the RNAi molecules and exposed to a series of electrical pulses. These interfere with the structure of the cell membrane, forming small hydrophilic pores in the membrane, which the RNAi molecules migrate through. This is a particularly cytotoxic method, and has a higher probability of cell death.
Sandwich ELISA kit information

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