siRNA is a chemically synthesized 21–23 nt double-stranded RNA that acts after direct cellular transfection. It has a rapid onset (24–72 h) but a transient duration (3–7 days), making it suitable for short-term experiments and high-throughput screening. shRNA is a hairpin RNA expressed from a DNA vector and processed intracellularly by Dicer into siRNA. It has a slower onset (48–96 h) but can achieve long-term silencing through stable plasmid expression or viral integration, making it suitable for generating stable knockdown cell lines.

Choose siRNA for short-term reversible knockdown and shRNA for long-term stable knockdown.

siRNA design follows several principles:
(1) The target sequence should be 21–23 nt long, ideally with 30–50% GC content;
(2) Avoid regions with strong mRNA secondary structure to maintain target-site accessibility;
(3) The thermodynamic stability of the sense-strand 3' end should be lower than that of the antisense-strand 5' end (AUM principle), promoting preferential loading of the antisense strand into RISC;
(4) Avoid high-frequency SNP sites and repetitive sequences;
(5) Perform genome-wide off-target analysis and exclude candidates whose seed region (positions 2–8) is highly homologous to the 3' UTR of non-target mRNAs.

We design the three-candidate package using established design principles and off-target analysis to maximize the likelihood that at least one of the three sequences is effective.

OPC (Oligonucleotide Purification Cartridge) is cartridge-based purification that removes short failed products and salts. It provides approximately 75% purity at lower cost and is suitable for routine cellular transfection experiments.

HPLC (high-performance liquid chromatography) purification provides purity ≥90%, removes failed products more thoroughly, and is suitable for applications requiring high purity, such as in vivo studies, transfection of sensitive cells, and quantitative experiments.

OPC is generally sufficient for transfection of standard cell lines such as HEK293 and HeLa. HPLC is recommended for primary cells, stem cells, animal studies, or data intended for publication.

After siRNA transfection, RISC loading and mRNA degradation occur within 24–48 hours. mRNA knockdown peaks at 48–72 hours, while protein-level knockdown typically peaks at 72–96 hours because existing protein must first be degraded. Knockdown lasts 3–7 days as siRNA is diluted by cell division, then gradually recovers.

For longer-lasting knockdown, use shRNA for stable expression or repeat siRNA transfection every 3–4 days.

siRNA transfection efficiency directly affects knockdown performance. Recommended measures include:
(1) Use Cy3- or FAM-labeled siRNA, or the Cy3-labeled negative control included in the package, to monitor transfection efficiency; target >80%;
(2) Optimize the transfection reagent and reagent-to-siRNA ratio; ZY FectEasy RNA Transfection Reagent may be used;
(3) Use healthy cells in logarithmic growth, at low passage, and free of mycoplasma contamination;
(4) Use a working siRNA concentration of 10–50 nM; higher concentrations increase off-target risk;
(5) For sensitive cells such as primary or stem cells, consider electroporation or viral delivery.

The three-candidate package includes a Cy3-labeled control for convenient monitoring of transfection efficiency.

Negative control (NC): an siRNA sequence unrelated to the target gene and designed not to target any gene. It verifies the specificity of the knockdown phenotype. If the NC also causes phenotypic changes, nonspecific effects such as off-target activity or transfection toxicity may be present. A Cy3-labeled NC also enables monitoring of transfection efficiency.

Positive control (PC): a validated siRNA targeting a housekeeping gene such as GAPDH or ACTB. It verifies that the entire experimental system, including the transfection reagent, cell condition, and assay method, is functioning properly. If the PC fails to achieve knockdown, troubleshoot the transfection conditions rather than the target sequence.