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siRNA Transfection: Experimental Procedures and Principles
siRNA is one of the most widely used forms of gene silencing (gene interference) in basic research, offering convenience and speed, and is extensively applied in studies of gene function across diverse eukaryotic organisms, as well as in drug target discovery and high-throughput drug screening. Standard siRNA products are lyophilized, ready-to-use reagents stored at −20°C. Target-specific sequences are designed to cover all genes in the human, mouse, and rat genomes, and, when delivered via chemical transfection, they are suitable for a wide range of cellular RNAi experiments. Moreover, by introducing specific chemical modifications to siRNAs, it is possible to achieve greater efficiency, specificity, and stability in in vivo applications.
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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). Synthesized siRNA enters the cell via endocytosis, after which a small fraction escapes the lysosome and translocates into the cytoplasm. Once in the cytoplasm, siRNA assembles with Dicer and TRBP to form the RISC complex. The RISC complex recruits AGO2; the guide strand is then degraded, while the passenger strand binds to an mRNA sequence that is fully complementary. Subsequently, AGO2 cleaves the mRNA, leading to its degradation.
siRNA Transfection Protocol (Below, we use a 24-well plate as an example; for transfection in other types of dishes, please consult Li Ji Bio.)
1. Cell inoculation
For adherent cells: The day before transfection, seed the cells at a density of 1–2 × 10^5 cells per well, aiming for a confluence of 60%–80% at the time of transfection.
For suspension cells: On the day of transfection, prior to preparing the transfection reagent–nucleic acid complex, wash the cells 1–2 times with PBS, resuspend them in 250 μL of Opti-MEM I serum-free medium, and seed the cells in a 24-well plate at a density of 60–80%.
2. Prepare the transfection reagent–siRNA complex (or transfection reagent–miRNA complex)
(1) For each well, add 2 μL of siRNA (20 μM, dissolved in DEPC‑treated water) to a 1.5 mL centrifuge tube, then add 2 μL of transfection reagent and mix thoroughly; incubate at room temperature for 3 minutes.
(2) Add 100 μL of Opti-MEM I serum-free medium to the above mixture, gently mix, and incubate at room temperature for 30 min to form the transfection reagent–nucleic acid complex.
(3) After 30 minutes, add 250 μL of Opti-MEM I serum-free medium to the above complex and gently mix.
3. Transfected cells
(1) Wash the cells with PBS 1–2 times, then add the above-mentioned 350 µL of transfection reagent–siRNA complex to each well.
(2) Incubate in a 37°C, 5% CO₂ incubator for 24–48 hours without replacing the culture medium; thereafter, you can assess the gene‑knockdown efficiency or proceed with downstream functional assays. Note: 500 μL of fresh complete culture medium may be added 12 hours post‑transfection.
siRNA transfection efficiency validation
Analysis of siRNA-mediated gene silencing: After transfection into cells, siRNA is processed within the cell by a series of enzymes to form an RNA-induced silencing complex, which recognizes and cleaves the target mRNA, triggering a degradation cascade in the host cell. This leads to the suppression of the expression of the intended gene product. Such effects are typically assessed from two perspectives.
• mRNA levels – QPCR analysis
The mechanism of siRNA action involves the degradation of target‑gene mRNA; therefore, the extent of mRNA degradation serves as direct evidence of siRNA‑mediated gene silencing efficiency. Typically, a reduction in target mRNA levels can be detected as early as 24 hours after siRNA transfection. mRNA knockdown efficiency can be assessed by extracting total cellular RNA, synthesizing full-length cDNA, and performing quantitative real‑time PCR.
• Protein levels – WB analysis
siRNA induces degradation of the target gene’s mRNA, thereby modulating the expression of the corresponding protein. However, the timing of protein-level analysis is influenced by factors such as the intracellular abundance, stability, and half-life of the target protein, as well as other regulatory mechanisms; consequently, the extent of protein downregulation does not necessarily correlate linearly with the reduction in mRNA levels. Typically, Western blotting is initiated 48 hours post-transfection, with multiple time points—72, 96 hours, or even longer—being sampled.
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