For DNA transfection, adherent cells should generally be at 50%–70% confluence. For RNA transfection, 30%–50% confluence is recommended. For suspension cells, use approximately 2×10⁶–4×10⁶ cells/mL. Cell density that is too high or too low can reduce transfection efficiency.

Serum-free medium or Opti-MEM is recommended when diluting nucleic acids and transfection reagent. After the complexes have formed, adding them to serum-containing medium usually does not affect efficiency. Antibiotics can increase cell permeability and reduce viability, so they should preferably be omitted from the transfection system, especially for sensitive cells.

For DNA transfection, confirm that cells are in logarithmic growth and free of contamination, check plasmid purity (A260/A280 ≥1.8) and endotoxin level, and optimize the DNA-to-reagent ratio, typically 1:1 to 1:2.5, and cell density. For RNA transfection, optimize siRNA and reagent concentrations, measure multiple time points from 24 to 96 hours, confirm that consumables are RNase-free, use PAGE-purified and desalted siRNA, and verify that annealing conditions are correct.

Common causes include poor cell condition, impure plasmid DNA or excessive endotoxin, too much transfection reagent, and delayed medium replacement. Reduce the reagent volume, shorten the time that complexes remain with the cells, replace the medium with fresh medium 4–6 hours after transfection, and determine whether silencing the target gene itself affects cell viability.

mRNA levels usually begin to decrease after 24 hours, while clear protein-level changes are generally seen after 48–72 hours. Measurements at 24, 48, and 72 hours are recommended. For DNA transfection, expression can usually be measured 24–40 hours after transfection.

Yes. ZY FectEasy RNA Transfection Reagent is suitable for delivering siRNA, miRNA mimics and inhibitors, saRNA, and other small-RNA molecules into cells. At low concentrations, it can achieve inhibition efficiencies above 90% in common cell lines such as HeLa, MCF7, and NIH-3T3, and above 80% in suspension cell lines such as K562 and THP-1.