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.