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?

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

What concentrations of mRNA are considered high,100ng/ul? Which buffer should I use to store mRNA? My mRNA activity still drops after 3 months at -80C and stored at 100ng/ul. I’ll appreciate any help-Marcia

Dear Marcia,

In general, 100 ng/uL is not considered a high concentration. We supply our mRNA at 1 mg/mL in 10 mM Tris-HCl, pH 7.5. That said, the mRNA sequence and structure can dictate its solubility. We recommend heating the mRNA for 15 min at 37°C to improve solubility. Long incubations at elevated temperatures should be avoided.

In regards to the diminishing activity of your mRNA, have you checked for degradation?  In addition to the solubility, your issues with mRNA activity may also result from RNA degradation over time. To combat degradation you should use RNase-free reagents and materials and use proper technique. Additionally, we suggest that you aliquot your RNA to limit freeze/thaw cycles.  A higher concentration may also improve stability however  could exacerbate your issues with solubility.

Please let us know if we can help you further.

Regards,

Brea

How do I concentrate mRNA?

mRNA can be concentrated using an Amicon® Ultra 30 kDa or 100 kDa size exclusion filter. These are available from EMD Millipore in various sizes. Simply add the mRNA to the filter and spin for a short time (~3-5 min) at the recommended speed. mRNA will be retained in the top chamber. Spin in brief rounds of centrifugation until the desired concentration is reached.  Note that over-concentrating the mRNA could lead to precipitation. Carefully remove the mRNA from the top chamber using a pipette. Calculate the actual concentration of the mRNA using a spectrophotometer or a Nanodrop™ device.

 

My experimental system contains RNases. How do I combat degradation?

For many applications it is desirable to increase the nuclease stability of the RNA. The most common approach is to substitute canonical bases with 2’-fluoro modified NTPs. Bacteriophage polymerases do not efficiently incorporate 2’ modified NTPs. However, selection strategies have been utilized to evolve polymerases that can incorporate 2’ modified NTPs. Researchers commonly substitute pyrimidine bases with 2’ fluoro modified bases when making RNAs for biological applications, such as aptamers.

What precautions should I take when working with mRNA and long RNA?

We recommend that surfaces are wiped down with RNase Zap® and disposable plasticware is used for all supplies and reagents that will contact RNA. Use RNase-free reagents and a fresh bottle of serum-free media for diluting RNA and lipids. Water can be made RNase free by treating with DEPC and autoclaving. Alternatively, you can purchase RNase-free reagents. If possible, dedicate a set of pipettes for RNA work and use barrier tips. Note that serum contains Rnases and will likely degrade your RNA very quickly.