Lipofectamine™ MessengerMAX™ Transfection Reagent

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?

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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.

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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.

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

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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.