I am trying to perform a RiboGreen Assay to calculate mRNA encapsulation efficiency. Triton buffer (1× TE buffer pH 7.5 with 2 % Triton X-100) was prepared and tested, but the concentration of Triton seems to be too high. How to determine the concentration of Triton X-100 in Triton buffer? Is there a correlation between lipid moles and Triton moles? Also, please let me know if there is another way to calculate the RNA encapsulation efficiency other than the RiboGreen Assay in the LNP experiment. Thank you.
You can measure the concentration of Triton X-100 in the buffer by its UV absorbance at about 280 nm, since neither Tris nor EDTA absorbs at that wavelength. According to one source, the absorbance of a 1% Triton solution at 280 nm with a 1-cm path-length is about 20. You can make a standard curve of your own Triton supply to be most accurate.
The higher the concentration of lipid present, the higher the concentration of Triton needed to dissolve it, but the precise relationship would have to be determined under your experimental conditions.
I would be concerned that the presence of Triton micelles and lipid membranes could both enhance the fluorescence of RiboGreen. I suggest you test this by leaving out the mRNA.
Another way to quantify the mRNA might be to run the extracted LNPs on agarose gels and stain for the mRNA band with a fluorescent dye, assuming all the lipids and detergents don't interfere with the electrophoresis.
7/15/22
Dear Yesseul,
Check this reference:
Journal: J AOAC Int.
. Mar-Apr 2016;99(2):396-400.J AOAC Int. Epub 2016 Mar 9.. Mar-Apr 2016;99(2):396-400.J AOAC Int
Direct UV Spectrophotometry and HPLC Determination of Triton X-100 in Split Virus Influenza Vaccine
Bojana Pavlović 1, Nataša Cvijetić, Luka Dragačević, Branka Ivković, Zorica Vujić, Vesna Kuntić
Affiliations expand
- PMID: 26960682
- DOI: 10.5740/jaoacint.15-0201
I hope this information helps you.
Bill Colonna Iowa State University, Ames, Iowa, USA [email protected]
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH