Lipid-Based Transfection—Troubleshooting

Freezing can alter the integrity and composition of the lipid particles. The performance of the reagent may be affected, and it may only work for the first week or so. It is safer to purchase a new vial to ensure function. 

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Below are possible reasons why you may see reduced viability following transfection, along with suggested solution.

1. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
   Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µl) ratio of 1:2to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine 2000 (µl) ratios from 1:0.5 to 1:5.
2. Possible Cause: Plasmid DNA preparation contains high levels of endotoxin
   Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPureNucleic Acid Purification Kits.
3. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
4. Possible Cause: Complexes were added to cells in serum-free medium
   Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
5. Possible Cause: Complexes not thoroughly mixed in growth medium
   Suggested Solution: Following addition of transfection complexes into medium, ensure that the plate or wells are thoroughly mixed to prevent concentration of DNA:transfection reagent complexes in the wells
6. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
7. Possible Cause: Antibacterial agents were used in growth medium during transfection
   Suggested Solution: Do not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity.
8. Possible Cause: Transfection reagent stored improperly
   Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.
9. Possible Cause: Cationic lipid reagent was oxidized
   Suggested Solution: Do not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides.
10. Possible Cause: Selection antibiotic added too soon
    Suggested Solution: When creating stable cell lines, allow at least 72 hr for cells to express the resistance gene before adding selective antibiotic.

In general, transfection efficiency will show some degree of variability between transfection experiments and among replicates in the same transfection experiment. For better reproducibility, keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. We recommend preparing one master mix of the DNA/lipid complexes for the number of transfections planned to reduce multiple pipetting errors. When adding your complexes, we recommend changing tips between wells since re-used tips could bring carryover, especially for the 96- or 384-well format with small-volume formats. To further minimize the effects of transfection variability on data analysis, consider co-transfecting an internal normalization reference control such as beta-galactosidase or luciferase with the expression plasmid. Below are possible reasons for why your transfection results are not reproducible, along with suggested solutions:

1. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
2. Possible Cause: Transfections performed at different cell confluencies, or at different DNA:transfection reagent ratios
   Suggested Solution: Transfection performance reproducibility is dependent on day-to-day consistency in cell splitting, plating and transfecting with a consistent protocol (same DNA:transfection reagent ratios). Different DNA preparations or media changes may also change transfection performance.

A common reason for seeing precipitate on your cells following lipid-based transfection is if there is excess EDTA or cationic lipid present. We recommend diluting the DNA in water or, if TE is preferred, use EDTA concentrations of <0.3 mM. Also ensure that concentrations of cationic lipid reagents do not exceed recommended amounts during complex formation. The presence or absence of this precipitate is not indicative of the transfection performance. 

Transfection with cationic lipids can produce light granular orange background fluorescence. The orange fluorescence is associated with the lipid/DNA complexes and is not related to GFP. This background varies depending on the cationic lipid reagent used and does not interfere with transfection results.  

Freezing can alter the integrity and composition of the lipid particles. The performance of the reagent may be affected and it may only work for the first week or so. It is safer to use a new vial. 

Yes, all of our lipid transfection reagents are stable at room temperature for months.

We also observed some toxicity in our initial experiments with mRNA. However, incorporating the proper 5’ UTR and 3’ UTR sequences into the template used for in vitro transcription quickly resolved it. Another key factor was the purity of the mRNA. Ensure that the OD 260/280 ratio is between 1.8 and 2.1. The quality of the mRNA can also be determined by running a small amount on a gel to check for the proper size. In some scenarios, it might be best to also incorporate chemically modified nucleotides in the transcription reaction. Another reason for toxicity may be a result of the cells themselves; if the cells are at too low a density then there will be significant toxicity (ideal viable cell density on the day of transfection is between 70 and 90% confluence).

We have seen this as well throughout our labs. Since we identified this issue, we have implemented a standard best practice of only utilizing cells for transfection between 5–20 passages, because at a low passage there is very low transfection efficiency and at a higher passage the optimal dose of transfection shifts. We have also implemented the standard use of a positive control within each transfection experiment to quantitatively determine and track efficiency from week to week.

There are reasons that can influence expression after transfection, but before troubleshooting all the possibilities, a transfection experiment with a positive control reporter and the Lipofectamine® MessengerMAX™ mRNA Transfection Reagent could be the solution. If this does not yield good results, it might be best to try an alternative delivery solution or different cells. However, if this gives acceptable results, the next step would be to try the mRNA of interest with the MessengerMAX™ reagent. If there are expression level concerns at this point, it might be the mRNA that is being used, and troubleshooting from this perspective might be needed. For example, some questions to ask would be: Is there a 5’ cap? Is there a poly(A) tail? Is the mRNA pure? Do I get a single band on a gel? Was the DNA template clean? 

Freezing can alter the integrity and composition of the lipid particles. The performance of the reagent may be affected and it may only work for the first week or so. It is safer to use a new vial. 

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Check to see if antibiotics were added to the medium during transfection, as this can cause cell death. Test serum-free media for compatibility with Lipofectamine® RNAiMAX Reagent.

This is not unusual; just vortex gently to resuspend the pellet, and use as recommended.

Check for column overload. We do not recommend using more than 15 to 30 mL per column. We recommend splitting the sample over 2 columns and re-spinning.