Purifying RNA by column chromatography.

Publisher Summary This chapter discusses chromatographic techniques that are used widely and that use readily available sorbents. Diethylaminoethyl (DEAE)-cellulose and DEAE-Sephadex are weak anion-exchange columns that have often been used to fractionate and purify RNAs. RNA binds to the column in low-salt buffers through ionic interactions between the phosphodiester backbone and the positively charged DEAE groups. Esterification of the hydroxyl groups of cellulose with benzoic acid or with a combination of benzoic and naphthoic acid substantially improves the resolution of DEAE-cellulose by increasing the hydrophobic characteristics of the column. Benzoylated DEAE-cellulose (BD-cellulose) and benzoylated, naphthoylated DEAE-cellulose (BND-cellulose) have similar properties to DEAE-cellulose, except that the binding interactions are generally stronger and that fractionation is more dependent on the secondary structure of the nucleic acid. The strength of the binding depends on the temperature, the divalent and monovalent cation concentration, and the pH. One of the major advantages of hydroxylapatite is its ability to discriminate between different nucleic acid structures. Hydroxylapatite also has a high binding capacity and is resistant to a wide range of temperature, pH values, and organic and inorganic solvents.