Lipofectamine™ MessengerMAX™ Transfection Reagent, 1.5 mL - FAQs

I am trying to troubleshoot our mRNA transfection process using Lipofectamine MessengerMAX Transfection Reagent. Could you tell me some reasons why we may not be seeing expression after transfection? We are using primary cells, so I am not sure if it is due to the difficulty in transfecting these cells or another cause.

There are reasons that can influence expression after transfection, but before troubleshooting all the possibilities, a transfection experiment with a positive GFP mRNA control (Tri-Link CleanCap EGFP mRNA, Cat. No. L-7201) 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?

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

We have seen quite a bit of variation in our mRNA transfection efficiency with Lipofectamine MessengerMAX Transfection Reagent, based on the number of passages prior to transfection. Do you have advice on the best passage number?

It is normal to observe some transfection efficiency differences between cell passages.To minimized this variability, we recommend using cells between 5-20 passages. Including a positive GFP mRNA control (Tri-Link CleanCap EGFP mRNA, Cat. No. L-7201) with every transfection experiment is helpful to quantitatively determine and follow any changes in transfection efficiencies from week to week.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I have been getting some toxicity in my mRNA transfections using Lipofectamine MessengerMAX Transfection Reagent. What would you recommend for minimizing toxicity?

Consider including appropriate 5' UTR and 3' UTR sequences into the template design to promote mRNA stability and acceptance by the cell. Use high purity in vitro transcribed mRNA purified with the MegaClear Transcription Cleanup Kit. Ensure A260/280 ratios are between 1.8 and 2.1. Check for mRNA quality and size by agarose gel electrophoresis. In some cases, consider incorporating chemically modified nucleotides in the transcription reaction. Cell number, mRNA, and lipid quantities may need to be adjusted. Ideal viable cell density on the day of transfection is between 70 and 90% confluence. For transfection optimization tips, please visit: thermofisher.com/transfectionsupport.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I accidentally left my lipid reagent at room temperature. Can I still use it?

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

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

When transfecting mRNA, does the packaging of the foreign RNA into vesicles reduce the accessibility of the mRNA for protein translation or is the mRNA all released in the cytoplasm? If packaged away, what percent is packaged vs. what percent is left to be available for translation? Is this observed with DNA plasmids?

We have not observed differences between how a cell packages an mRNA payload versus a DNA payload for the purpose of delivery. Transfection involves complex formation between a liposome and mRNA, which create lipoplexes that are taken up by the cell via endocytosis. The liposome protects the mRNA during this process and also assists in endosomal escape, which releases the mRNA into the cytoplasm of the cell. The mRNA is immediately available for translation with the ribosome. In vitro transcribed mRNA may be prepared using the mMESSAGE mMACHINE T7 Ultra Transcription Kit, which incorporates a 5' ARCA cap and a 3' poly(A) tail to mimic endogenously transcribed mRNA.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the best transfection reagent to use to transfect mRNA into cells/tissue in a low-pH environment? I have tried most of the mRNA transfection reagents on the market, and they do not work.

The best reagent to transfect mRNA into cells is Lipofectamine MessengerMAX Transfection Reagent because it has been shown to further protect the mRNA molecules from degradation and has been shown to deliver the highest amount of mRNA into a wide variety of cell types. Please visit this page (http://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-messengermax.html) for more information. For tissues or small animal models, the best reagent is Invivofectamine 3.0 Transfection Reagent, which is based on a nanoparticle technology that encapsulates and protects the payload for delivery. It can be used on cells in vitro as well as injected systemically via intravenous route or via direct injection (i.e., muscle, tumors, heart, brain). More information can be found here (http://www.thermofisher.com/us/en/home/life-science/rnai/introduction-to-in-vivo-rnai/invivofectamine-reagent/invivofectamine-reagent-data.html?cid=fl-invivofectamine).

In addition, mRNA can also be synthesized with chemically modified nucleotides to improve stability and further minimize degradation. A positive control mRNA (e.g., GFP mRNA) is very helpful to optimize transfection techniques.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is transfection with mRNA faster? What are standard incubation times?

Yes, transfection with mRNA results in faster and more immediate translation of protein and therefore, faster expression. We visually see expression of GFP in some cell lines as fast as 90 minutes after transfection. Additionally, transfection of mRNA with Lipofectamine MessengerMAX Reagent also provides prolonged duration of expression (GFP expression lasting for 5 days post-transfection), due to its ability to protect mRNA from degradation during transfection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are there specific medium requirements when using mRNA for transfection? Also, would I have to change the medium after transfection?

No, there are no specific medium requirements or restrictions when using mRNA for transfection. The only recommendation is to ensure that there is no serum or antibiotics present during the complexation process of the lipid and mRNA within the transfection protocol. We use Opti-MEM I Reduced Serum Medium in our transfection protocol. The delivery of mRNA does not require a medium change after transfection. Our common practice is to not change medium within the first 24 hours following transfection, so as to minimize handling of cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

We currently use DNA for transfection, but what are the advantages for mRNA transfection? Is it cell line-specific?

There are many advantages to using mRNA vs. DNA for transfection, and they are cell line-specific:

Higher transfection efficiency:
If you are working with a difficult-to-transfect cell type, where DNA transfection yields less than 30% efficiency, transfecting with mRNA can provide up to 80% transfection efficiency. During DNA transfection, the DNA encounters many hurdles before reaching the nucleus before undergoing the molecular processes that ultimately lead to protein expression. The DNA-lipid complexes first fuse to the membrane, enter the cell by endocytosis, DNA is released from the endosomes and and enters the nucleus. In the nucleus, the DNA is transcribed to mRNA, exported from the nucleus, and is finally translated into a protein in the cytoplasm. During mRNA transfection, the mRNA is ready for immediate translation to protein in the cytoplasm. However, if you are satisfied with the level of transfection achieved for a given cell line, there is no need to switch to mRNA.

Footprint-free method with no risk of genomic integration:
mRNA transfection is transient, does not enter the nucleus, or pose a risk of integrating with the cellular host DNA. mRNA transfection is currently being researched for possible vaccine replacement and disease model development.

Additionally, transfection of mRNA with Lipofectamine MessengerMAX Reagent provides higher efficiency in a wider range of cell types (e.g., primary neurons, primary hepatocytes, primary keratinocytes, primary fibroblasts, iPS cells, hNSC, mESC, Raw 264.7, SH-SY-5Y, HT-29). This is a result of its ability to deliver the highest amount of mRNA independent of the cell model being used.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use Lipofectamine MessengerMAX Transfection Reagent to deliver plasmid DNA?

Yes, Lipofectamine MessengerMAX Transfection Reagent can deliver plasmid DNA in combination with mRNA (i.e., in CRISPR); however, Lipofectamine 3000 Transfection Reagent is best optimized for plasmid DNA delivery and superior DNA transfection performance.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How easy is it to use Lipofectamine MessengerMAX Transfection Reagent?

Lipofectamine MessengerMAX Transfection Reagent has a very simple one-tube protocol (http://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_MessengerMAX_man.pdf). The mRNA can either be transcribed using the mMESSAGE mMACHINE ULTRA T7 Transcription Kit or can be purchased ready-to-use (e.g., GeneArt CRISPR Nuclease mRNA, Cat. No. A25640).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Where can I purchase a GFP mRNA positive control to use with Lipofectamine MessengerMAX Transfection Reagent?

We have validated the TriLink EGFP mRNA Control (Cat. No. L-6101) in-house. As this was discontinued, the alternative is Tri-Link CleanCap EGFP mRNA (Cat. No. L-7201).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Does the Lipofectamine MessengerMAX Transfection Reagent kit include reagents for in vitro transcription of mRNA?

Although it does not, we recommend the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Cat. No. AMB1345) to generate capped and tailed mRNA for highest mRNA stability.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

When do you recommend using the Lipofectamine MessengerMAX Transfection Reagent?

The Lipofectamine MessengerMAX Transfection Reagent is specifically designed for transfection of mRNA into cells. It offers up to 5X the transfection efficiency of DNA transfection reagents in neurons and a broad spectrum of primary cells. This is possible because our novel lipid nanoparticle technology is optimized to deliver the highest amount of mRNA possible without the nuclear entry step that is required with DNA. The added advantage is that protein expression is faster with no risk of genomic integration. Additionally, up to 10X higher cleavage efficiency can be achieved using GeneArt Platinum Cas9 Nuclease for CRISPR gene editing applications.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Which transfection reagent should I use for delivering my gene of interest into stem cells?

We recommend using Lipofectamine Stem Transfection Reagent for delivery of DNA, mRNA and co-transfection (siRNA and plasmid DNA) in a wide variety of stem cells. Lipofectamine Stem Transfection Reagent also delivers Cas9-gRNA ribonucleoproteins for gene editing applications. For more information on the transfection efficiency and versatility of the reagent, please visit: https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-stem-transfection-reagent.html

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Do you offer a transfection reagent for transfection of mRNA?

Yes, Lipofectamine MessengerMAX Transfection Reagent is our best reagent for mRNA delivery and shows up to 5Xs the transfection efficiency of DNA transfection reagents in neurons and primary cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What controls do you recommend that I use when performing a transfection?

To ensure optimal transfection success, we recommend including a positive transfection control and additional controls to confirm cell health and reagent quality.

For DNA transfection, we recommend using the pJTI R4 Exp CMV EmGFP pA Vector (Cat. No. A14146). For siRNA transfection, we recommend using either the BLOCK-iT AlexaFluor Red Fluorescent Control (Cat. No. 14750100) or Silencer Select GAPDH Positive Control siRNA (Cat. No. 4390850). For protein transfection, we recommend co-transfecting with an EmGFP mRNA such as the Tri-Link CleanCap EGFP mRNA (Cat. No. L-7201).

Cell health and reagent quality controls:
- Cells only
- Cells + DNA or RNA or protein only
- Cells + lipid reagent only
- Cells + Opti-MEM only
- Cells + positive control

Find additional tips, troubleshooting help, and resources within ourTransfection Support Center.

What points should I consider to achieve optimal transfection with a lipid-based transfection reagent?

Here are some points to consider:

1. Select the lipid reagent that is likely to result in highest transfection efficiency for your cell type, payload, and application. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent.
2. Optimize both lipid reagent and DNA quantities. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum may contain components that could interfere with complex formation. We recommend using Opti-MEM I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, but the efficiency of complex formation may not be as high as with Opti-MEM I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be between 50% to 80% confluency at the time of transfection (please refer to specific reagent manual for details). Cells should be in the mid-log growth phase. For better consistency and reproducibility of results between transfection experiments, accurately count your cells with either a hemocytometer or the Countess II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Confirm that the promoter and/or enhancer (any gene regulatory sequences) of the transfected DNA is compatible with the target cell type.
7. Do not use a cationic lipid reagent that has been frozen or stored at temperatures below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

For additional tips ,please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

What is the stability of your transfection reagents?

Our transfection reagents are shipped under ambient conditions and should be stored at 4 degrees C immediately upon receipt. We guarantee the performance of the product, if stored and handled properly, for one year from date of shipment unless otherwise stated on the tube label or COA. We do not recommend freezing transfection reagents, as this usually decreases transfection performance.

Please see this white paper (http://tools.thermofisher.com/content/sfs/brochures/cms_103226.pdf) on ambient shipping of Lipofectamine transfection reagents.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why do I see cytotoxicity after performing transfection with Lipofectamine 2000? Can you please help?

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.

I am getting very low transfection efficiency with Lipofectamine 2000. Can you please provide some troubleshooting tips?

Below are possible reasons why you may be getting low transfection efficiency, along with suggested solutions:

1. Possible Cause: Plasmid DNA, siRNA, or transfection reagent diluted in media containing serum or complexes formed in the presence of serum
   Suggested Solution: Use serum-free medium for dilutions of plasmid DNA, siRNA, and transfection reagents. Note: we recommend using Opti-MEM | Reduced Serum Medium (Cat. No. 31985-062)to dilute Lipofectamine 2000 and DNA before complexing.
2. 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 1:2 to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine2000 (µL) ratios from 1:0.5 to 1:5. If using a different transfection reagent, please consult the product manual.
3. Possible Cause: Not enough plasmid DNA used for dilution or complex formation
   Suggested Solution: Verify concentration using a second method or check the DNA for degradation. Determine DNA concentration by performing A260/A280 readings on a spectrophotometer or by using the Quant-iT DNA Assays Kits (Q33130, Q33120).
4. Possible Cause: Plasmid DNA or siRNA used in transfection has degraded or is of poor quality
   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 HiPure Nucleic Acid Purification Kits.
5. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
6. 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.
7. Possible Cause: Inhibitors were present in medium
   Suggested Solution: Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the growth medium or in the medium used to prepare DNA:transfection reagent complexes.
8. Possible Cause: Problems with assay used to measure efficiency or expression
   Suggested Solution: Use a reporter gene to measure transfection efficiency. A reporter gene control allows you to confirm expression.
9. Possible Cause: Promoter-enhancer on vector is not recognized by the cell type
   Suggested Solution: Verify that the promoter-enhancer on your vector construct is compatible with the target cell type.
10. 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.
11. 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.


Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

How do I perform a dose-response curve or kill curve?

The dose-response curve is a valuable tool to determine cell toxicity when exposed to various concentrations of antibiotic. The amount of selective antibiotic required to select for resistant cells varies with a number of factors, including cell type and type of antibiotic. We recommend performing a dose-response curve every time a new antibiotic (or a different brand) or a different cell line is used.

Experimental outline of dose-response curve assay:

1.Plate cells in a number of wells such that they are 25–30% confluent. This means that the cells are still dividing and hence will respond well to the antibiotic.
2.Dilute the antibiotic being tested to a broad linear concentration of the recommended range in growth medium.
3.Remove the growth medium from the cells. Apply the antibiotic-containing medium to the respective wells, leaving one set of wells empty. To these wells, add growth medium that does not contain the antibiotic.
4.Culture cells under proper growth conditions (change the medium every 3–4 days to get rid of dead cells and add fresh medium containing antibiotic) and observe the cells daily. At 10–14 days, assess the number of viable cells in each well. (This time period depends upon the antibiotic being tested; antibiotics such as Geneticin, Hygromycin, and Zeocin take about 3 weeks to kill cells, so waiting for 10–14 days would be ideal. However, for Blasticidin, which kills cells in about 2 weeks, waiting for 7–10 days would be sufficient.) To do this, aspirate the medium, wash the cells with phosphate-buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
5.Plot the number of viable cells against the antibiotic concentration. This curve is the dose-response curve or kill curve. The lowest concentration of the antibiotic that kills all the cells in the chosen time period is then used for the stable selection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of viral transduction over transfection?

Transfection does not work for certain cell types such as non-dividing cells, whereas viral transduction works for dividing as well as non-dividing cells, such as neuronal cells that are hard to transfect.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of lipid-mediated transfection over calcium phosphate-mediated transfection?

The main advantage of lipid-mediated transfection is the higher transfection efficiency that can be achieved with cell types that cannot be transfected using calcium phosphate. Calcium phosphate is prone to variability due to its sensitivity to slight changes in pH, temperature, and buffer salt concentrations. Calcium phosphate may also be cytotoxic to many cell types, especially primary cells. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What types of molecules can be transfected using your lipid-based transfection reagents?

Our cationic lipid transfection reagents are used to transfect DNA (plasmids or oligonucleotides), siRNA (or miRNA), mRNA, or proteins. DNA delivered may be in the form of plasmids, cosmids, or even YAC clones as large as 600 Kb. Please refer to the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent based on cell type and application.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is there a place where I can find references from other researchers who have used your reagents?

Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Where can I find cell line-specific transfection protocols?

Cell line-specific transfection protocols can be found here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html). If you do not find a cell line-specific protocol or if the transfection does not perform as expected, we recommend optimizing the conditions described in the product manual. Successful transfection depends on the cell type, amount of lipid, cell health, passage number, and cell density at the time of transfection. Each of these factors may differ slightly from lab to lab and may require additional optimization of the protocol to achieve the same result. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For more troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport).

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Why would the expression level of my gene in transiently transfected cells be greater than those that are stably transfected?

Expression in transiently transfected clones is typically higher because transiently transfected cells can have up to hundreds of copies of the plasmid containing the gene of interest. Stably transfected clones usually harbor 1-2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why are my transfections not reproducible?

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.

I am working with well sizes different from those specified in your protocol. How do I scale up or scale down my transfection reaction?

Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details. For well or plates sizes not listed in the scaling table, calculate the total surface area and estimate the -fold difference from the 24-well. Use this -fold difference to adjust for reagent volumes, payload quantities, and seeding densities.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use the same amount of any transfection reagent for different cell lines?

No.The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.

The protocol that is supplied with the product will provide you with an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For additional troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport)

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are cell density (% confluency) and passage number important considerations for transfection?

Yes, cell density is an important parameter in influencing transfection efficiency. If the seeding density is too low, some cytoxicity may be observed. If the cell density is high, lower than expected transfection efficiency may be observed. Both issues may be easily resolved by either descreasing or increasing the quantity of complexes added to the culture. We recommend using Lipofectamine 3000 since it shows the best flexibility for variable seeding density without showing cytotoxicity issues and maintains high protein expression. Lipofectamine 3000, Lipofectamine 2000, and Lipofectamine LTX/PLUS provide excellent transfection efficiencies at confluencies between 70 and 90%. Some toxicity may be observed at lower confluencies but may be alleviated by decreasing quantity of complexes or removing the complexes after 4-6 hours incubation and refreshing the media. Lipofectamine RNAiMAX works best at confluencies between 60 and 80%.

Passage number may affect transfection experiments. We recommend consistent splitting and plating of cells. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20-30 passages. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen. Please refer to the Gibco Cell Culture Basics handbook (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html) for proper guidelines for culturing and passaging cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What recommendations do you have for selecting a transfection reagent?

Choose the best reagent by cell type and application by using the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What are the different methods available for transfection?

There are many transfection methods available to deliver plasmids, DNA fragments, oligos, siRNAs, mRNA, or proteins for a wide range of research and drug discovery applications. A review of the pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I deliver single-stranded oligonucleotides with Lipofectamine MessengerMAX Transfection Reagent?

If you are delivering a single-stranded oligonucleotide on its own, we recommend using Lipofectamine RNAiMAX Transfection Reagent. However, Lipofectamine MessengerMAX Transfection Reagent can be used for genome editing purposes when Cas9 mRNA is co-delivered with a sgRNA and a single-stranded or double-stranded donor template.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do I use Lipofectamine MessengerMAX Reagent to deliver both my GeneArt Cas9 mRNA and IVT gRNA together?

Lipofectamine MessengerMAX Reagent may be used for both single gRNA delivery or multiplexing (gRNA for multiple targets) purposes with high transfection efficiency when used with GeneArt Cas9 mRNA. For a detailed protocol, please refer to the GeneArt Cas9 mRNA manual (https://www.thermofisher.com/order/catalog/product/A25640?ICID=search-a25640). We recommend using either the Invitrogen GeneArt Genomic Cleavage Detection Kit (Cat. No. A24732) or Invitrogen GeneArt Genomic Cleavage Selection Kit (Cat. No. A27663) for mutant analysis.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Which cell types can be used with Lipofectamine MessengerMAX Transfection Reagent?

Lipofectamine MessengerMAX Reagent demonstrates low toxicity and high transfection efficiency for all cell types (easy or difficult-to-transfect, primary, and stem cells).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

When would I use Lipofectamine MessengerMAX Reagent over other transfection reagents?

Lipofectamine MessengerMAX Regent is an excellent reagent for co-delivery of Invitrogen GeneArt CRISPR Cas9 mRNA with in vitro transcribed gRNA for geneome editing purposes. Lipofectamine MessengerMAX Reagent has the added benefit of flexibly delivering short dsDNA or HDR templates (0.5-1 kb) which can be ordered through the Invitrogen GeneArt Strings services.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the Invitrogen Lipofectamine MessengerMAX Transfection Reagent?

Lipofectamine MessengerMAX Transfection Reagent is an animal origin-free transfection reagent, especially formulated for the delivery of mRNA, small RNA (eg.,CRISPR IVT gRNA, siRNA, or miRNA), and short dsDNA or HDR templates (0.5-1 kb). Lipofectamine MessengerMAX Reagent is an excellent reagent choice for CRISPR-mediated genome editing applications.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.