Category Archives: Transfection

Are the Requirements for Using CleanCap mRNA Different Than ARCA mRNA?

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The requirements for using CleanCap mRNA are no different than using ARCA mRNA. You can use your standard electroporation or transfection conditions. There are several advantages with using a CleanCap mRNA. The translational efficiency far exceeds that of a standard ARCA capped mRNA. You may want to re-purpose your solar eclipse glasses when viewing your cells after EGFP translation!

And just like with ARCA, you can submit your open reading frame to TriLink for custom mRNA transcription and we will do the rest!

How much mRNA is needed to transfect cell cultures in 24-well plates?

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There are typically two avenues for transfection of mRNA depending on cell type:

1. For adherent cells, use a transfection reagent such as MessengerMax (Invitrogen), mRNA TransIT (Mirus) or mRNA-In (MTI-GlobalStem).

a. For a 24-well plate format, use a ratio of 500 ng mRNA:1 ul transfection reagent in a total volume of 50 ul complexed mRNA/lipid per well. This is a good starting point and is described in this protocol. Diluting your working stock of mRNA down to 100 ng/ul works well to establish manageable volumes for mastermixes.

b. We’ve only used TransIT and mRNA-In in-house – from our experience these reagents give a dose response of FLuc activity from between 100 ng to 400 ng. Therefore, a minimum starting amount of 100 ng is sufficient. Use 100 ng mRNA: 1ul reagent in a complexed final volume of 50 ul per well.

c. Ratios for other plate formats need to be optimized according to the manufacturer’s instructions.

2. For cells in suspension, such as CD34+ cells, electroporation has traditionally been the mode of mRNA delivery. However, transfection reagents have advanced and some are able to transfect cells in suspension reliably, such as the transfection reagent mRNA-In (MTI-GlobalStem).

a. For electroporation, an instrument from Lonza is recommended based on advice from a trusted collaborator.

b. Use 1-15 ug of mRNA per million cells in a 100 ul volume.

We recently purchased eGFP mRNAs (L-6101 and L-6402) as controls for mRNA delivery experiments. I have transfected HEK293 and DU145 cells using lipofectamine 3000 and PEI (5:1 weight ratio) and am unable to see GFP expression (by FACS) after 7 or 24 hours. By FACS (looking at cy5) the delivery looks good. I have tried both the labeled (L-6402) and unlabeled (L-6101) side by side and transfected 100 or 200 ng/well (96-well plate) into cells at about 50% confluency. Control transfections using these cell lines with a CMV-eGFP plasmid produce almost off-scale GFP expression when measured by FACS. Is there something wrong with my protocol and what is the timing and level of GFP expression I should expect in comparison to a plasmid transfection?

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Dear Scott,

Thank you for your inquiry. I would suggest checking for mRNA degradation. Make sure you are using serum free reagents (ie Optimem) that are rigorously RNAse free. We use special pipets for mRNA and for example do not do minipreps or maxipreps with these pipets. RNasezap can be used to clean work surfaces and pipets. FACS signal from the Cy labeled RNA does not ensure that there was good delivery since the RNA could simply be trapped in an endosome. This experiment should work well in the 293 cells, we have no experience with the other cell line. Additionally, you make want to experiment with the timing. Though I would expect to see EGFP expression at your indicated time points, many factors influence expression and half-life. We and others typically see peak expression between 12-18 hours.

Regards,
Brea

I don’t see any expression after transfection. What should I do?

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We suggest validating the mRNA in an easily transfected cell line, such as HEK-293 cells. You may also want to include a GFP plasmid as a positive control. Note that while the GFP plasmid will provide confirmation of transfection, is does not verify delivery to the correct cellular compartment. The target compartment for the plasmid is the nucleus and the target compartment for the mRNA is the cytoplasm.

Which transfection reagent should I use?

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Several companies offer transfection reagents. Our collaborators have used TransIT®-mRNA Transfection Kit (Mirus),  Stemfect™ (Stemgent), mRNA-In™ (MTI-GlobalStem), RmesFect™ Transfection Reagent (Oz Biosciences), and Lipofectamine™ RNAi Max (Life Technologies) with success. We suggest testing a matrix of transfection reagents and varying ratios of RNA to transfection reagent. Just as with plasmids and oligonucleotides, the optimal transfection procedure will need to be determined empirically. Some cell types are intrinsically easy to transfect (e.g. HEK-293 cells ~97%). Efficient delivery to other cell types, such as some primary cells can be very challenging.