Transfection

RNA transfection is the introduction of RNA into cells by means other than viral infection. Transfection of RNA is similar to transfection of plasmids. It is used to express the encoded protein in cells, study gene expression and regulation, and study gene function. RNAi studies use siRNA to determine the effects of knocking down genes. Transfection of RNA can be done quickly. 

Transfection of RNA into cells is performed to express its encoded protein transiently and to study the kinetics of decaying RNA. Transfection of RNA is usually performed on cells that do not divide. 

RNA can be extracted from cells following lysis or synthesized using free nucleotides by chemically or enzymatically employing an RNA polymerase to create the DNA template. Like DNA, RNA is transferred to cells using electroporation, microinjection, and lipids.  

When RNA encodes proteins, the transfected cells could translate the RNA into encoded protein. When the RNA is a regulator RNA (such as a miRNA), RNA could trigger different cell changes (such as knockdown mediated by RNAi). 

Incorporating RNA molecules into lipid nanoparticles was crucial for the development of viable RNA vaccines, overcoming many technical challenges in delivering the molecules into human cells. 

RNA molecules that are smaller than 25nt (nucleotides) are mostly not detected by the innate immune system, which is activated by longer RNA molecules. Most body cells have proteins that are part of the innate immune system, and after exposure to long RNA molecules from outside, they trigger signals that lead to inflammation.  

Therefore, short RNA can be transfected repeatedly without causing non-specific reactions, transfecting cells repeatedly with even a small amount of long RNA can cause cell death unless both the innate immune system and the transfection are suppressed. 

What Are The Methods of Transfection? 

For RNA transfection, several biological, physical, and chemical methods are available. The most effective methods of transfection utilize non-viral methods (electroporation and calcium phosphate) as well as liposome-based transfection in which cargo molecules are delivered to cell membranes without causing permanent damage. 

Chemical methods neutralize DNA’s negative charge, allowing it to be absorbed more readily. While the physical transfection procedure involves electroporation, microinjection, and biolistic particle delivery. 

Advantages of RNA Transfection 

Transfecting mRNA offers several advantages over DNA transfection. There is no risk of high transfection efficiency that is cyclic in the host genome and no need for immune-inducible vectors or variable and fast expression. The other advantages of mRNA transfection: 

• By using mRNA transfection, you can insert any number of mRNAs in cells, thus preventing the overexpression of genes.  
• mRNA functions without a nucleus, so it is more efficient. 
• The level of expression of transfected DNA depends on the strength of the enhancer carried by the tissue or host cell promoter.  
• Contrary to DNA transfection, it is possible to modify the expression level by altering the quantity of mRNA and the mRNA transfection rate.  
• mRNAs are immediately expressed after transfection, as it avoids the transfer to the nucleus and the transcription processes.  
• As transcription home (population of microRNAs) is a useful tool to transfect, this is a real challenge to achieve in the case of DNA transfection. 

Because of this, the transfecting of RNA is drawing attention to its therapeutic potential. However, it is essential to recognize that matured mRNA is composed of five important structures the cap 5′ untranslated area (5’UTR), the open reading frame (ORF), 3 untranslated regions (3’UTR), the poly-A tail) and undergoes nucleoside changes that affect the translation of the transcript.  

So, the plasmid used to perform in-vitro transcription has to be developed, taking into account all aspects that influence stability and translation efficiency. Although handling mRNA requires more care, there are plenty of applications for switching from DNA transfection to mRNA transfection.