Extracellular RNA Isolation from Biofilm Matrix of Pseudomonas aeruginosa

Background

Biofilm matrix provides resistance to different influences (antibacterial drugs, disinfectants, mechanical forces) and creates environment for coordinated regulation of different processes (Svenningsen, 2018). RNA presents in extracellular biofilm matrix and forms the main cross-linking elastic copolymer of RNA-DNA (Seviour et al., 2019). Treatment of biofilms with RNases resulted in significant loss of biofilm mass and underline importance of RNA for maintenance of biofilm integrity (Lee et al., 2019). At the same time sources and roles of RNA in biofilm matrix remain poorly explored.

The use of solutions with high ionic strength allows separation of extracellular biofilm matrix without subjecting embedded cells to lysis (Chiba et al., 2015). At the same time further workflow of RNA purification might be significantly affected with high salt concentration (i.e., phase separation during phenol-chloroform extraction). Another important aspect of such method of separation includes relatively large volume of the dissolved biofilm matrix, so there is a need of efficient RNA precipitation for further purification steps. The method of RNA isolation from biofilm matrix described by Seviour et al. (2019) includes matrix separation with high ionic liquids, for example, 40% (v/v) 1-ethyl-3-479 methylimidazolium acetate (EMIM Ac): 60% (v/v) N,N-dimethyl acetamide (DMAc) and Perchloric acid (70%) and further steps of RNA isolation by chromatographic separation, but these steps include some harsh conditions (i.e., incubation at 55 °C for 16 h and long centrifugation steps). It is not easily applicable in molecular biology laboratories. We imply idea of using high ionic liquid from both studies, implement LiCl precipitation to concentrate and redissolve RNA and clean up with inexpensive silica columns adopted from (Yaffe et al., 2012). Finally, we offer a simple, fast, and inexpensive method of extraction of extracellular RNA (exRNA), moreover intracellular cell RNA (cRNA) might be purified from the same biofilm sample. Protocol utilizes common laboratory reagents and equipment and enables to isolate extracellular RNA suitable for further procedures. Such a method may be applicable for studying the molecular mechanisms of biofilm formation and maintenance.