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Cell Transfection Principles and Complete Protocol

🚨 Failure Case Library (11) + Submit your own case

critical
Massive cell death after transfection — reagent toxicity is too high
Cells become rounded and detached after transfection; CCK-8 shows a sharp drop in viability vs. mock-transfected control.
💡 4 · ✓ 4
critical
Almost no fluorescence signal — transfection essentially failed
Under the fluorescence microscope, no GFP signal is visible; Western blot also shows essentially no target protein expression.
💡 5 · ✓ 5
severe
Very few cells are fluorescent — transfection efficiency is extremely low
Microscopy shows scattered positive cells; flow cytometry confirms positivity rate is very low (< 5 %).
💡 5 · ✓ 5
severe
Very Low Transfection Efficiency
Transfection efficiency is significantly lower than expected with poor gene expression
💡 7 · ✓ 9
severe
Empty/negative control also shows fluorescence (high false-positive background)
Untransfected or empty-vector wells show a clearly green/red signal — you can't tell if the target plasmid is really expressing.
💡 4 · ✓ 4
severe
Bright fluorescence but Western blot shows very low expression
GFP fluorescence is obvious under microscope, but the corresponding band on Western blot is faint or near-invisible.
💡 5 · ✓ 5
moderate
mRNA Transfection Toxicity
Cell toxicity observed during mRNA transfection experiments with MessengerMAX reagent
💡 5 · ✓ 7
moderate
Very high replicate-well variation (poor reproducibility)
Parallel wells show very different fluorescence intensity / positive-cell rates; data fluctuates wildly between replicates.
💡 4 · ✓ 4
moderate
Co-transfection ratio imbalance — uneven dual-plasmid expression
Of two co-transfected plasmids, only one expresses strongly; the fraction of double-positive cells is low.
💡 5 · ✓ 4
moderate
Transfections Not Reproducible
Transfection efficiency shows high variability between experiments and among replicates
💡 5 · ✓ 7
moderate
Abnormal fluorescence localization (aggregates / wrong compartment)
Fluorescence forms aggregates inside cells, or localizes to a compartment different from the expected native pattern.
💡 4 · ✓ 4
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