title: Purification of pre-miR-29 by arginine-affinity chromatography.
abstract: Recently, differential expression of microRNAs, in patients with Alzheimer's disease (AD) suggests that they might have key regulatory roles in this neurodegenerative disease. Taking into account this fact, several studies demonstrated that the miR-29 is significantly decreased in AD patients, also displaying abnormally high levels of β-site APP-cleaving enzyme 1. Thus, RNA biochemical or structural studies often require a RNA sample that is chemically pure and biologically active. The present work describes a new affinity chromatography method using an arginine support to specifically purify pre-miR-29 from other Rhodovulum sulfidophilum small RNA species. Nevertheless, in order to achieve higher efficiency and selectivity, it is essential to characterize the behavior of pre-miR-29 binding/elution. Thus, three different strategies based on increased sodium chloride (280-500mM), arginine (25mM) or decreased ammonium sulfate (2-0.1M) stepwise gradients are described to purify pre-miR-29. In this way, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (96.5 and 56.7%, respectively) was obtained. Moreover, the quality control analysis revealed high integrity in pre-miR-29 preparations as well as high purity (90 and 98%, respectively), demonstrated by the scarce detection of proteins. This improved method takes advantage of its simplicity, significant cost reduction, due to the elimination of some complex operations, and speed for large-scale purification of pre-miRNAs suitable for biochemical and structural studies.
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors: Patrícia
authors: P
authors: Pereira
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors: Ângela
authors: Â
authors: Sousa
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors: João
authors: J
authors: Queiroz
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors: Ilídio
authors: I
authors: Correia
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal; CNC-Center of Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal.
authors: Ana
authors: A
authors: Figueiras
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal. Electronic address: fani.sousa@fcsaude.ubi.pt.
authors: Fani
authors: F
authors: Sousa
mesh_terms: D001120:Arginine / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D035683:MicroRNAs / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009994:Osmolar Concentration; D041903:Rhodovulum / Q000737:chemistry / Q000235:genetics; D013696:Temperature
chemical_list: D035683:MicroRNAs; D001120:Arginine
keywords: Alzheimer's disease; Arginine-affinity chromatography; Multiple interactions; RNA purification; Rhodovulum sulfidophilum; pre-miR-29