461990 Ultrafiltration of Ribonucleic Acids
Ribonucleic acid from Torula yeast was used as a model RNA. The RNA was suspended in Tris-EDTA buffer, with concentrations measured using the Quant-iT RiboGreen RNA Assay. RNA integrity was examined by Agarose Gel Electrophoresis with the gels visualized using GelStar Nucleic Acid Gel Stain. Ultrafiltration experiments were performed in a stirred cell apparatus using Ultracel composite regenerated cellulose membranes with 100 kD nominal molecular weight cutoff (pore size of approximately 10 nm).
RNA transmission increased with increasing filtrate flux, similar to behavior observed previously with plasmid DNA. This is likely due to the elongation of the RNA molecules in the converging flow field entering the membrane pores. However, the RNA transmission decreased with increasing ionic strength, which is exactly the opposite of the behavior observed with DNA. This effect was quite pronounced. For example, at a flux of 0.1 cm/s, the RNA transmission decreased from more than 60% in the TE buffer to less than 2% in the same buffer with 10 mM NaCl. This large effect of ionic strength on RNA transmission is likely due to changes in the secondary and / or tertiary structure of the RNA due to strong intramolecular interactions. These results provide important insights into the possible use of ultrafiltration for RNA purification, including the separation between DNA and RNA under different ionic conditions.