Denaturing reversed phase liquid chromatographic separation of non-coding ribonucleic acids on macro-porous polystyrene-divinylbenzene resins

• We examine the LC separation of RNAs on macro-porous polystyrene-divinylbenzene resins.
• The method separates RNAs of 20–8000 nucleotides with high sensitivity.
• The separation occurs essentially according to the molecular size of RNA.
• The method allows for the separation of various RNAs from biological sources.
• The isolated RNAs can be characterized by nanoflow LC–tandem mass spectrometry.

The ability of denaturing ion-paired reversed phase LC to separate RNA was assessed using macro-porous polystyrene-divinylbenzene resins as the stationary phase. Using the three stationary phases with different pore size and a mobile phase containing phosphate, we separated RNAs of 20–8000 nucleotides with extremely high sensitivity, e.g., 50 pg for an RNA 20 nucleotides in length, S/N = 5. The method was used to separate non-coding RNAs obtained from biological sources and is suited for use with direct MS-based chemical characterization.