I am preparing stock solutions of RNase A from powder form and the preparation details mention that it is necessary to prepare it with multiple buffers and boil it for 10 minutes. Is this necessary? Could it affect staining with PI if used without eliminating DNase activity? The downstream application is FACS cell cycle analysis.
Cell cycle analysis by flow cytometry using PI staining is based on the determination of cellular DNA. In this process, cells are permeabilized and RNase A is employed to degrade cellular RNA that would otherwise interfere with DNA analysis. By definition, DNase activity in the RNase A would be detrimental in this analysis, due to degradation of cellular DNA, which is essentially the analytical target in this analysis. DNase free RNase A, purified by chromatography, and suitable for this application, is available from several suppliers. In case of less pure RNase A products, inactivation of DNase activity would be necessary and it requires pH adjustment to avoid thermal precipitation and the loss of RNase A activity. Lastly, the presence of DNase activity in the less pure RNase A products is probably due to the fact that they are both present in and isolated from the bovine pancreatic material.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques