title: Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus.
abstract: Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
authors.forename: C O
authors.initials: CO
authors.lastname: Beltrame
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
authors.forename: M F
authors.initials: MF
authors.lastname: Côrtes
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
authors.forename: P T
authors.initials: PT
authors.lastname: Bandeira
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
authors.forename: A M S
authors.initials: AM
authors.lastname: Figueiredo
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods / Q000592:standards*; D018441:Biofilms / Q000254:growth & development*; D004587:Electrophoresis, Agar Gel; D006460:Hemolysin Proteins / Q000378:metabolism; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification; D011786:Quality Control; D012329:RNA, Bacterial / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D048348:Reverse Transcription; D013211:Staphylococcus aureus / Q000235:genetics* / Q000502:physiology
chemical_list: D006460:Hemolysin Proteins; D012329:RNA, Bacterial