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Comprehensive Guide: Procedures for Transient Cell Transfection
source：QiDa technoligy views：136 time：2026-07-06
What is transient transfection?
It refers to the process where exogenous nucleic acids (such as plasmid DNA or mRNA) are introduced into eukaryotic cells without integrating into the host genome, and are only briefly expressed intracellularly (typically for 24–96 hours) at high levels, followed by gradual dilution or degradation during cell division, leading to progressively diminishing expression until complete disappearance.
Common methods for transient expression:
Liposome transfection (e.g., Lipofectamine series)
Calcium phosphate co-precipitation method
Electroporation
Mediated by viral vectors (e.g., adenovirus, non-integrative type)
Instant expression is primarily used for:
Rapid validation of gene function
Analysis of protein overexpression
Promoter activity detection
RNA interference (siRNA/shRNA) experiment
Reports gene expression assays (e.g., luciferase assays)
1.Cell culture
Cell status: In the logarithmic growth phase, confluence rate 70–90%
Cell preparation: Use cells with low passage numbers (<20 passages). Prior to infection, test for Mycoplasma contamination; if negative, proceed with experimentation. If contaminated, eliminate Mycoplasma first and then culture with a Mycoplasma-eliminating reagent for two generations for optimal infection efficacy
Culture medium: Replace with antibiotic-free complete medium 2–4 hours before transfection (antibiotics may reduce transfection efficiency)
2. Plasmid Preparation:
Plasmid purity: A260/A280 = 1.8–2.0
Endotoxin-free: recommended concentration 0.5–5 μg/μL; dissolved in sterile water or TE buffer solution
3. Reagent Preparation:
Transfection reagents (e.g., Lipofectamine 3000, PEI, FuGENE, etc.)
Opti-MEM, or serum-free culture media (for dilution)
Transfection procedure steps (using a 6-well plate as an example)
| step | Operation Content |
| 1.Bed board | Cells were inoculated 18–24 hours prior to transfection, ensuring a confluence rate of ≥70% at the time of transfection. |
| 2.Dilute the plasmid | Take 2.5 μg of plasmid DNA, dilute it with 125 μL of Opti-MEM, and mix thoroughly. |
| 3.Dilution Reagent | Take 3.75 μL of Lipofectamine 3000 and dilute it with 125 μL of Opti-MEM; alternatively, take 125 μL of Opti-MEM plus 2.5 μL of P3000 for dilution, then store for use. |
| 4.Mixing | Combine the diluted plasmid with the transfection reagent in equal volumes, and incubate at room temperature for 10–15 minutes to form a DNA-liposome complex. |
| 5.Sample addition | Add 250 μL of the complex dropwise into the cells, and gently shake to mix. |
| 6.Cultivation | Continue cultivation in a 37°C incubator with 5% CO₂. |
| 7.Media change | Change the culture medium every 4–6 hours using complete medium. |
Reference dosage for transient cell cultures using different orifice plates (using Lipofectamine 3000 as an example)
| Culturing vessels | Cell population | DNA (μg) | reagent(μL) |
| 96-well plate | 1–2×10⁴ | 0.1 | 0.15 |
| 24-well plate | 1–2×10⁵ | 0.5 | 0.75 |
| 6-well plate | 2–4×10⁵ | 2.5 | 3.75 |
| 10-cm dish | 1–2×10⁶ | 7.5 | 11.25 |
Pay attention to details for a one-step transfection:
·Cell density is critical: excessive density (>95%) reduces transfection efficiency, while insufficient density compromises cell viability.
·Antibiotic-free conditions: Avoid using double antibiotics during transfection to minimize cytotoxicity.
·Plasmid quality: Endotoxins significantly impair transfection efficiency and induce cell death.
·Serum effects: While some reagents are compatible with serum, most liposome-based methods require dilution in serum-free medium.
·Complex incubation time: Do not exceed 20 minutes to prevent aggregation and precipitation.
·Toxicity control: Reduce reagent volume or shorten medium change intervals if severe cell death occurs.
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