title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues.
abstract: Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides and important components of extracellular matrices and cell surface glycan layers such as the endothelial glycocalyx. The GAG family includes sulfated heparin, heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate, and non-sulfated hyaluronan. Because relative expression of GAGs is dependent on cell-type and niche, isolating GAGs from cell cultures and tissues may provide insight into cell- and tissue-specific GAG structure and functions. In our objective to obtain structural information about the GAGs expressed on a specialized mouse glomerular endothelial cell culture (mGEnC-1) we adapted a recently published GAG isolation protocol, based on cell lysis, proteinase K and DNase I digestion. Analysis of the GAGs contributing to the mGEnC-1 glycocalyx indicated a large HS and a minor CS content on barium acetate gel. However, isolated GAGs appeared resistant to enzymatic digestion by heparinases. We found that these GAG extracts were heavily contaminated with RNA, which co-migrated with HS in barium acetate gel electrophoresis and interfered with 1,9-dimethylmethylene blue (DMMB) assays, resulting in an overestimation of GAG yields. We hypothesized that RNA may be contaminating GAG extracts from other cell cultures and possibly tissue, and therefore investigated potential RNA contaminations in GAG extracts from two additional cell lines, human umbilical vein endothelial cells and retinal pigmental epithelial cells, and mouse kidney, liver, spleen and heart tissue. GAG extracts from all examined cell lines and tissues contained varying amounts of contaminating RNA, which interfered with GAG quantification using DMMB assays and characterization of GAGs by barium acetate gel electrophoresis. We therefore recommend routinely evaluating the RNA content of GAG extracts and propose a robust protocol for GAG isolation that includes an RNA digestion step.
authors.affiliation: Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Jasper J
authors.initials: JJ
authors.lastname: van Gemst
authors.affiliation: Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Markus A
authors.initials: MA
authors.lastname: Loeven
authors.affiliation: Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Mark J J
authors.initials: MJ
authors.lastname: de Graaf
authors.affiliation: Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Jo H M
authors.initials: JH
authors.lastname: Berden
authors.affiliation: Department of Nephrology and Einthoven Laboratory for Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Ton J
authors.initials: TJ
authors.lastname: Rabelink
authors.affiliation: Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Cornelis H
authors.initials: CH
authors.lastname: Smit
authors.affiliation: Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
authors.forename: Johan
authors.identifier: http://orcid.org/0000-0001-7843-5918
authors.initials: J
authors.lastname: van der Vlag
mesh_terms: D000464:Alginates / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000737:chemistry; D003871:Dermatan Sulfate / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D020723:Glucuronic Acid / Q000737:chemistry; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000302:isolation & purification / Q000592:standards; D006497:Heparan Sulfate / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry; D007632:Keratan Sulfate / Q000737:chemistry; D007668:Kidney / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D051379:Mice; D012313:RNA / Q000302:isolation & purification*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism; D013154:Spleen / Q000378:metabolism*
chemical_list: D000464:Alginates; D002809:Chondroitin Sulfates; D003871:Dermatan Sulfate; D020723:Glucuronic Acid; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D006603:Hexuronic Acids; D006820:Hyaluronic Acid; D007632:Keratan Sulfate; D012313:RNA