title: Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilum.
abstract: The present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025 μg of plasmid DNA, with a heat-shock of 2 min at 35 °C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182 μg/L, after 40 h of bacterial growth, and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 h. At the end of the fermentation, it was verified that almost 87 % of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics.
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: Augusto Q
authors: AQ
authors: Pedro
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal.
authors: Joana
authors: J
authors: Tomás
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal.
authors: Cláudio J
authors: CJ
authors: Maia
authors: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal.
authors: João A
authors: JA
authors: Queiroz
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. fani.sousa@fcsaude.ubi.pt.
authors: Fani
authors: http://orcid.org/0000-0001-9996-2194
authors: F
authors: Sousa
mesh_terms: D003470:Culture Media / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D041903:Rhodovulum / Q000235:genetics / Q000378:metabolism*
chemical_list: D003470:Culture Media; C532045:MIRN29a microRNA, human; D035683:MicroRNAs
keywords: Extracellular production; RNAi technology; Recombinant human pre-miR-29b; Rhodovulum sulfidophilum; Transformation efficiency