SiRNA is currently one of the most commonly used forms of gene silencing (gene interference) in basic research, which is convenient and fast. It is widely used in gene function research, drug target discovery, and drug screening in various eukaryotes. Conventional siRNA products are ready to use reagents in freeze-dried powder form, stored at -20 ° C. Design specific targets that cover all genes of the human, mouse, and rat genomes, and utilize chemical transfection techniques suitable for conducting various cellular RNAi experiments. In addition, by performing special chemical modifications on siRNA, more efficient, specific, and stable in vivo experiments can be achieved.
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Principle of siRNA transfection
The mechanism by which siRNA degrades mRNA in cells is similar to that of miRNA (as shown in the figure above). The synthesized siRNA enters the cell through endocytosis, and then a small amount of siRNA can escape into the cytoplasm through lysosomes. After entering the cytoplasm, siRNA forms a RISC complex with Dicer and TRBP. The RISC complex recruits AGO2, and then the sense chain is degraded. The siRNA antisense chain binds to the perfectly complementary mRNA sequence, and then AGO2 cleaves the mRNA, ultimately causing mRNA degradation.
SiRNA transfection experimental steps (using a 24 well plate as an example, transfection in other culture dishes can be consulted with Li Ji Biology)
1. Inoculate cells
For adherent cells: the day before transfection, plate the cells at a density of 1-2x10 ^ 5 cells per well to achieve a density of 60% -80% during transfection.
For suspended cells: On the day of transfection, before preparing the transfection reagent nucleic acid complex, wash the cells 1-2 times with PBS, resuspend the cells in 250 μ L Opti MEM I serum-free medium, and plate the cells in a 24 well plate with a cell density of 60-80%.
2. Prepare transfection reagent siRNA complex (or transfection reagent miRNA complex)
(1) For each well of cells, take 2 μ L of siRNA (20 μ M, dissolved in DEPC water) and add it to a 1.5mL centrifuge tube. Mix 2 μ L of transfection reagent with siRNA and incubate at room temperature for 3 minutes.
(2) Add 100 μ L Opti MEM I serum reducing medium (serum-free) to the above mixture, gently mix well, and let it stand at room temperature for 30 minutes to form a transfection reagent nucleic acid complex.
(3) After 30 minutes, add 250 μ L of LOpti MEM I serum reducing medium (serum-free) to the above complex and mix gently.
3. Transfect cells
(1) Wash the cells 1-2 times with PBS and add 350 µ L of transfection reagent siRNA complex to each well of cells.
(2) Cultivate in a 37 ℃, 5% CO2 incubator for 24-48 hours without the need to replace the culture medium, and then the gene interference effect or subsequent functional experiments can be tested. Note: 500 μ L of complete culture medium can be supplemented 12 hours after transfection.
SiRNA transfection efficiency validation
Analysis of siRNA silencing effect: After siRNA transfection into cells, siRNA forms RNA induced silencing complexes under the action of a series of enzymes in the cell, recognizes and cleaves target gene mRNA, induces host cell degradation reactions against these mRNAs, and thus inhibits the expression of target gene proteins in the cell. Generally, detection can be carried out from two aspects.
MRNA level - QPCR detection
The mechanism of action of siRNA is that it causes the degradation of target gene mRNA, so the level of mRNA degradation is a direct proof of siRNA silencing efficiency. Generally, the decrease in target mRNA levels can be detected 24 hours after siRNA transfection. Total RNA extraction, full-length cDNA synthesis, and fluorescence quantification experiments can be used to detect the knockdown efficiency of mRNA.
Protein level - WB detection
SiRNA induces the degradation of target gene mRNA, thereby affecting the expression of target proteins. However, the time for protein level detection is influenced by factors such as the expression level, stability, half-life, and even other regulatory factors of the target protein in the cell. The magnitude of protein level decrease may not necessarily be linearly proportional to the decrease in mRNA level. Generally, WB detection begins 48 hours after transfection, and multiple sampling points are taken after 72, 96 hours, or even longer.
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