Journal: Experimental & Molecular Medicine

Article Title: Proteins are a source of glycans found in preparations of glycoRNA

doi: 10.1038/s12276-025-01575-1

Figure Lengend Snippet: a A schematic representation of a purification strategy to rescue glycosylated molecules from the flow-through of silica columns used for the purification of RNase-treated RNA samples. The flow-through of the first silica column loading step is saved and transferred to an Amicon concentrator column. b In-gel fluorescence detection of Ac 4 ManNAz-labeled glycans rescued from the flow-through of RNase-treated HeLa RNA. The lanes show an input control (‘Input’), samples incubated with or without RNase A/T1 before column loading (‘Load’), after elution (‘Elu’) and the Amicon-concentrated flow-through (‘FT’). An untreated RNA (‘Input’) was subjected to Amicon concentration as a control. Identical samples were analyzed by agarose gel electrophoresis (left and middle panel) and SDS–PAGE (right panel). A representative gel from three independent experiments is shown ( n = 3). c In-gel detection of labeled glycans obtained from silica column purifications in the presence of intact RNA or increased levels of isopropanol. RNA extracted from metabolically labeled 3T3 cells was incubated with or without RNase A/T1. After digestion, samples were split in three and then subjected to a silica column purification with three different conditions during the precipitation step : one tube received one volume of isoproanol, the second received 5 µg of intact RNA and one volume of isopropanol and the third received two volumes of isopropanol. The samples were purified in parallel and analyzed via agarose gel electrophoresis. The samples before the silica column run are shown as controls (‘Input’). A representative gel of two independent experiments is shown ( n = 2).

Article Snippet: The labeling medium consisted of 25 ml of Dulbecco’s modified Eagle medium or Roswell Park Memorial Institute 1640 medium with supplements as stated above and 100 μM Ac 4 ManNAz (Jena Bioscience), 100 μM GalNAc (Sigma) and 10 μM D-Gal (Roth).

Techniques: Purification, Fluorescence, Labeling, Control, Incubation, Concentration Assay, Agarose Gel Electrophoresis, SDS Page, Metabolic Labelling