The process of cellular uptake and intracellular trafficking of transfected RNA depends on the method used for transfection. Here’s a general overview:

1. Cellular Uptake

• Lipofection: In this method, RNA is encapsulated in lipid vesicles (liposomes). These liposomes fuse with the cell membrane, releasing the RNA into the cell. The fusion can happen either at the cell surface or in endosomes, which are membrane-bound compartments inside the cell.
• Electroporation and Nucleofection: These methods use an electric field to create temporary pores in the cell membrane, allowing the RNA to enter. In nucleofection, the RNA is delivered directly into the nucleus.
• Microinjection: In this method, RNA is directly injected into the cell, bypassing the cell membrane.
• Viral vectors: Viruses have evolved to efficiently enter cells. The specific method of entry depends on the type of virus, but generally, the virus binds to specific receptors on the cell surface, leading to the uptake of the virus (and the RNA it carries) into the cell.

2. Intracellular Trafficking

Once inside the cell, the fate of the transfected RNA depends on its type:

• Messenger RNA (mRNA): After entering the cell, the mRNA needs to reach the ribosomes, where it will be translated into protein. Ribosomes can be found in the cytosol and on the endoplasmic reticulum. mRNA can be translated immediately after entering the cell.
• Small interfering RNA (siRNA) and microRNA (miRNA): These types of RNA need to be incorporated into the RNA-induced silencing complex (RISC). This happens in the cytoplasm. Once in the RISC, these RNAs can bind to their target mRNAs and cause their degradation or prevent their translation.
• Long non-coding RNA (lncRNA) and circular RNA (circRNA): The function of these types of RNA can vary, and they can act in different parts of the cell, including the nucleus and the cytoplasm.

It’s important to note that one of the challenges of RNA transfection is ensuring that the RNA reaches the right location in the cell. For instance, while mRNA can be translated immediately upon entering the cell, if it gets trapped in endosomes after lipofection, it may not be able to reach the ribosomes. Various strategies are being researched to improve the efficiency of intracellular delivery and trafficking of transfected RNA.