How can we determine the amount of two different RNA (example: siRNA and mRNA) when co-encapsulated in a single nanoparticle? We have been trying with ribogreen but it gives the total RNA estimate!
To specifically determine the amount of two different RNA molecules (e.g., siRNA and mRNA) co-encapsulated in a single nanoparticle, you can use fluorescently labeled probes or primers that are complementary to each RNA type. This technique allows you to differentiate between the two RNA molecules and quantify them separately, overcoming the limitation of total RNA estimation.
Hello Sadhanna,
as Nouhaila mentioned a qPCR based approach would be best.
In "Critical evaluation of quantification methods for oligonucleotides formulated in lipid nanoparticles" (2018) I used also qPCR to determine the amount in a nanoparticle in comparison to dye based techniques.
In my current publication, "Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma (2023)", in table 2 you can see how to design a specific primer for reverse transcription and the forward and reverse one for qPCR. We made the design after Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., et al.(2005). "Real-time quantification of microRNAs by stem-loop RT-PCR", Kramer, M. F. (2011). "Stem-loop RT-qPCR for miRNAs" and Varkonyi-Gasic, E., and Hellens, R. P. (2011). "Quantitative stem-loop RT-PCRfor detection of microRNAs".
Take a try and add six miRNA specific bases to our design by hand and test it. Normally it should work very well and then you can differentiate your siRNA and mRNA (or you wanted to write miRNA?). In the "Identification of extremely...." there is also a LNA probe described and you can use two dyes to differentiate both in one PCR run.
Best wished Volker