title: Histidine affinity chromatography-based methodology for the simultaneous isolation of Escherichia coli small and ribosomal RNA.
abstract: Research on RNA has led to many important biological discoveries and the improvement of therapeutic technologies. In particular, there is a great focus on small RNA and ribosomal RNA owing to their key functions in the cell, which make them excellent therapeutic targets. Although the study of these RNA classes is progressing, some limitations have been found regarding the use of suitable techniques that are able to produce and isolate biologically competent and chemically stable RNA. To address this, we have developed a novel histidine affinity chromatography-based isolation methodology for small and ribosomal RNA molecules. The new procedure involves three main steps: (1) cell lysis with guanidinium buffer, (2) RNA primary isolation with ammonium sulfate precipitation and (3) histidine affinity chromatography to specifically purify small RNA and ribosomal RNA from other Escherichia coli impurities (genomic DNA and proteins). The RNA quality assessment revealed that both RNA species were obtained with a high recovery, integrity and purity. The potential of this method to achieve a reproducible RNA isolation with appropriate quality has been demonstrated and it should have broad application in the structural, biophysical and biomedical investigation of systems involving RNA components.
authors: CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors: Rita
authors: R
authors: Martins
authors: João António
authors: JA
authors: Queiroz
authors: Fani
authors: F
authors: Sousa
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000302:isolation & purification*; D015203:Reproducibility of Results
chemical_list: D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D006639:Histidine