What should be paid attention to during siRNA transfection experiment?

In the process of transfection experiment, we should pay attention to the following points: a. purification of siRNA: confirm the size and purity of SiRNA before transfection. In order to obtain high-purity siRNA, it is suggested that the excess nucleotides, oligonucleotides, protein and salt ions in the reaction should be eluted or removed by 15%20% acrylamide gel.

Note that chemically synthesized RNA usually needs to be purified by running gel electrophoresis (that is, PAGE gel purification)

B. avoid RNase pollution: a small amount of RNase will lead to the failure of siRNA experiment. Because RNase is ubiquitous in the experimental environment, such as skin, hair, everything touched by hands or exposed to the air, it is very important to ensure that every step of the experiment is not polluted by RNase.

C. Healthy cell culture and strict operation ensure the repeatability of transfection: generally, the transfection efficiency of healthy cells is high, and the lower passage times can ensure the stability of cells used in each experiment. In order to optimize the experiment, it is suggested to transfect cells below 50 generations, otherwise the transfection efficiency will decrease obviously with time.

D. Avoid using antibiotics: Ambion recommends avoiding using antibiotics from cell implantation to 72 hours after transfection. Antibiotics can accumulate toxins in infiltrated cells. When siRNA is transfected, some cells and transfection reagents need serum-free conditions. In this case, both normal medium and serum-free medium can be used for comparative experiments to obtain the best transfection effect.

E. Selection of appropriate transfection reagent: According to the different preparation methods of siRNA and target cell types, it is very important to select a good transfection reagent and optimize the operation for the success of siRNA experiment.

F. Optimize transfection and detection conditions through appropriate positive control: housekeeping gene is a good positive control for most cells. Different concentrations of positive control siRNA were transferred to target cells (also suitable for experimental target siRNA), and after 48 hours of transfection, the reduction level of control protein or mRNA relative to untransfected cells was calculated. Excessive siRNA can lead to cytotoxicity and even death.

G. optimizing the experiment by labeling siRNA: fluorescently labeled siRNA can be used to analyze the stability and transfection efficiency of siRNA. Labeled siRNA can also be used as intracellular localization of siRNA and double labeling experiment (using labeled antibody) to track the cells introduced with siRNA during transfection, so as to combine transfection with down-regulation of target protein expression.