The phenomenon of RNA interference (RNAi) is triggered by double-stranded RNA that is homologous to the target gene sequence and is a post-transcriptional silencing process of sequence-specific genes that widely exists in organisms. The endonuclease Dicer in the cytoplasm cleaves dsRNA into siRNA consisting of 21-25 nucleotides, and then the siRNA combines with proteins in the body to form the RNA-induced silencing complex RISC. RISC specifically binds to the homologous region of mRNA expressed by exogenous genes and cleaves the mRNA at the binding site. After being cleaved, the fragmented mRNA is immediately degraded, thereby blocking the post-transcriptional gene silencing mechanism of the corresponding gene expression.
RNAi technology also provides innovative methods and approaches for new drug development and disease treatment, and has extremely broad application prospects. Transfection of siRNA is the most commonly used and important transfection of RNAi technology.
(1) Before transfection, ensure that siRNA has been purified by PAGE and desalted. High-purity siRNA or miRNA helps to achieve higher transfection efficiency and ensures that siRNA/miRNA gene silencing expression will not affect cell viability.
(2) First transfection: If you are transfecting your cell line for the first time, it is recommended to try several siRNA transfection reagent compound concentrations and change the siRNA concentration in the range of 0.014μg/mL~0.70μg/mL. Adjust dose levels based on staining results.
(3) Selection of siRNA transfection reagent diluent: There are no special requirements for the medium. Complete medium can be used for dilution. Reduced/serum-free medium is not required.
(4) Transfection cell density: It is recommended to conduct transfection at about 70%. Usually gene silencing analysis is performed 24 hours after transfection. Healthy cell cultures and strict procedures ensure reproducible transfections. Generally, healthy cells have higher transfection efficiency, and a lower passage number can ensure the stability of the cells used in each experiment. It is recommended to use cells below 50 passages for transfection.
(5) Positive control selection: For most cells, GADPH-siRNA is a better positive control. Different concentrations of positive control siRNA or experimental siRNA are transferred into target cells. 24 hours after transfection, use the internal reference gene such as β-actin mRNA as a control to calculate the reduction level of target mRNA relative to untransfected cells.
(6) Avoid RNase contamination: Trace amounts of RNase can cause siRNA experiments to fail. Since RNase is ubiquitous in the experimental environment, such as skin, hair, all items touched by bare hands or items exposed to the air. Therefore, please ensure that every step of the experiment is free from RNase contamination. It is recommended to use commercial enzyme-free experimental consumables.
(7) Effect evaluation: After cell transfection, there are differences in the efficiency of different cells and siRNA. The best time to observe the effect of siRNA at the mRNA level is 24 to 48 hours after transfection, and the best time to observe the effect of siRNA at the protein level is 48 to 72 hours after transfection. siRNA interference is non-linear, and the half-lives of different genes vary greatly. When measuring the siRNA interference effect for the first time, it is recommended to measure at multiple points to determine the best measurement time point.