siRNAs can be produced as follows:
Chemical synthesis: The chemical synthesis of siRNAs allows the production of 21 to 22 base-pair siRNA oligonucleotide duplexes. Chemically synthesis is a relatively simple and quick way to generate siRNAs.
In vitro transcription (IVT): IVT uses T7 RNA polymerase to produce siRNAs.
Endogenous expression: The endogenous expression of siRNAs produces short hairpin RNAs (shRNAs)delivered to cells via plasmids, viral or bacterial vectors.
RNAi, as a tool, allows for studying gene function in cell cultures. Introducing siRNAs or short hairpin RNAs (shRNAs) into cells specifically silences the expression of a target gene. RNAi used in cell cultures allows studying the effects of gene knockdown on cellular processes, such as cell growth, differentiation, and death.
How are siRNAs delivered into cells?
The delivery of chemically synthesized siRNAs to mammalian cells is possible through several strategies, such as direct conjugation to cell-surface binding ligands, encapsulation into lipids, and electroporation. However, plasmid-based shRNA vectors are usually delivered with the help of lipids or electroporation, and infection allows the delivery of virus-based vectors into cells.
Three principal delivery methods are possible:
During transfection, siRNAs or shRNAs are introduced into cells using chemical transfection, electroporation, or lipofection. However, transduction introduces nucleic acids into cells with the help of a viral vector.
- Delivery of naked siRNA.
- Delivery using siRNA packaged in lipids.
- Delivery as siRNA-conjugates.
RNAi has several advantages over other gene silencing methods, such as antisense oligonucleotides and gene knockout mice.
RNAi is more efficient and specific than antisense oligonucleotides, and it does not require the generation of knockout mice.
RNAi is also a relatively quick and easy method to perform.
RNAi has been used to study various biological processes, including cell growth, differentiation, death, and signaling. It has also enabled the development of new therapeutic approaches for multiple diseases, such as cancer and viral infections.
Benefits of using RNAi in cell cultures:
- Specificity: RNAi allows silencing the expression of a single gene without disturbing the expression of other genes.
- Efficiency: RNAi can efficiently silence gene expression, even for genes expressed at low levels.
- Versatility: RNAi allows silencing gene expression in various cell types, including primary cells, immortalized cell lines, and stem cells.
- Speed: RNAi enables silencing gene expression within a few hours or days.
Challenges of using RNAi in cell cultures:
Off-target effects: siRNAs and shRNAs can sometimes target unintended genes, leading to off-target effects.
Delivery: Delivery of siRNAs and shRNAs into cells can be challenging, especially for primary and stem cells.
Stability: Endogenous enzymes in cells can degrade siRNAs and shRNAs, limiting their duration of action.
Despite these challenges, RNAi is a powerful tool for studying gene function in cell cultures. With careful design and optimization, RNAi can generate reliable and reproducible results.
" Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides".