Something About siRNA Transfection
What is RNA interference?
RNA interference (RNAi) refers to the phenomenon of silencing gene expression due to the degradation of the mRNA when a double stranded RNA (dsRNA) homologous to the coding region of the endogenous mRNA is introduced into the cell. This phenomenon occurs at the post-transcriptional level and is also known as post-transcriptional gene silencing (PTGS). The antisense strand of small interfering RNA (siRNA) produced by exogenous dsRNA entering the cell and various nucleases form the RNA-induced silencing complex (RISC). RISC has the role of binding and cleaving mRNA to mediate the process of RNA interference. siRNA is one of the most popular fields of life science research and the most promising new drug development field in the future. siRNA transfection experiments have gradually become a common method in life science research.
Key points for siRNA transfection
- siRNA purification
The concentration and purity of siRNA are very important for transfection experiments. To obtain high-purity siRNA, it is recommended to remove excess nucleotides, small oligonucleotides, proteins and salt ions from the reaction by glass fiber binding, elution or by 15-20% acrylamide gels.
Note: Chemically synthesized RNA usually needs to be purified by running PAGE gel electrophoresis.
- Avoiding RNase contamination
Trace amounts of RNase will cause siRNA transfection experiments to fail. Since RNases are ubiquitous in the experimental environment, such as skin, hair, all objects touched with bare hands or exposed to the air, etc., it is very important to ensure that each link in the experiment is free from RNase contamination.
- Reproducibility of transfection ensured by healthy cell cultures and rigorous handling
Generally, healthy cells are transfected more efficiently. In addition, lower passage numbers ensure the stability of cells used in each experiment. In order to optimize the experiment, it is recommended to use transfected cells under 50 passages, otherwise the transfection efficiency of cells will decrease significantly over time.
- Selecting the right transfection reagent
According to the different siRNA preparation methods and target cell types, the selection of good transfection reagents and optimized operations are critical to the success of siRNA experiments. Such as BOC Sciences' siRNA Transfection Reagent, specifically designed for siRNA in vivo transfection without RNase, DNase and endotoxin.
Difference between siRNA transfection and DNA transfection
|Because siRNA has only two dozen base pairs, siRNA transfection is suitable for small RNA molecules.
|Transfection reagents for transfected DNA are targeted to relatively large plasmid DNA.
|Requirements for cytotoxicity
|siRNA experiments require minimal cytotoxicity of the transfection reagents.
|Cytotoxicity has little effect on overexpressed DNA transfection experiments.