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!
Suggested protocol for labeling Aminoallyl FLuc mRNA
This protocol is compatible for use with most water-soluble NHS esters*. If using a non-water soluble NHS-ester, different processing will be required. Precipitation in alcohol, such as isopropanol or ethanol, should be considered to remove excess NHS ester and any hydrolysis products. Multiple precipitations may be required.
*This protocol has been tested at small scales (100 ug) using Aminoallyl FLuc mRNA and Cyanine 5. Scales larger than 10 mg may require modifications to the protocol. The protocol has been designed to minimize mRNA degradation during the labeling process.
Salt exchange of Aminoallyl FLuc mRNA
1. Concentrate desired amount of Aminoallyl FLuc mRNA with 3K centrifugal filter. Reduce volume to ~0.25X of original.
2. Add 100 mM NaHCO3 to achieve total volume of 1.25X of original.
3. Repeat steps 1-2 at least three times. Add enough 100 mM NaHCO3 at final step to achieve 1X original volume of product. Concentration should be at ~ 1 mg/mL.
4. Add 1X volume 100 mM NaHCO3 in H20. Mix gently by inverting several times.
5. Add 2X volume 2 mM NHS ester solution in DMSO. Mix gently by inverting several times.
6. Incubate 90 min at room temperature. If label is fluorescent, make sure to shield from light.
7. Add ~0. 35X volume 4M hydroxylamine in H20. Mix gently by inverting several times.
8. Incubate in dark for 15 min at room temperature. If label is fluorescent, make sure to shield from light.
9. Concentrate ~3X with 10K centrifugal filter device.
10. Add back 3X volume H2O.
11. Repeat steps 9-10 at least 6 times.
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.
Under certain conditions mRNA may precipitate, particularly at high concentrations. Try heating the mRNA for 15 min at 37°C to improve solubility. Long incubations at elevated temperatures should be avoided.
mRNA can have solubility issues at high concentrations. This is particularly true if the sequence is codon optimized, as with TriLink’s Cas9 mRNA. Heating the mRNA for 15 min at 37°C may improve solubility. Long incubations at elevated temperatures should be avoided.
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.
We have assessed activity of our stocked EGFP in a variety of cell types, including HEK-293, RAW 264.7 and BJ Fibroblast cells. Additionally, we have expressed other stocked mRNA in HEK-293, CHO, BJ Fibroblasts, CEM and primary human CD34+ cells.
Our standard purification consists of two silica column purification steps. If you are interested in alternative purification methods, please contact us.
TriLink’s stocked mRNA products have been optimized for expression in mammalian cells and organisms. Activity in non-mammalian cells (i.e. flies, fish and worms) has not been evaluated. If you achieve expression with one of our stocked mRNA products in cells from other species, please share your results with us.