Babu Raman1, Courtney M. Johnson1, Dawn M. Klingeman1, Miguel Rodriguez Jr1, Steven D. Brown1, Jonathan R. Mielenz1, Brian H. Davison1, Larry Feinberg2, Christopher D. Herring2, and David Hogsett2. (1) Biosciences Division and BioEnergy Science Center, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, (2) Mascoma Corp., 67 Etna Road, Suite 300 Lebanon, NH 03766, Lebanon, NH 03766
The study of microbial physiology and associated phenotypes through array-based expression analysis requires isolation of high quality RNA. Here we report the extraction of high yield, high quality total RNA (RNA Integrity Number, RIN >8.0) from the anaerobic, thermophilic, Gram-positive bacterium, Clostridium thermocellum in the presence of large amounts of solid substrates (up to 500mg of crystalline cellulose or pretreated biomass, corresponding to 33-50g/L fermentation substrate load). Industrial ethanol production processes require operating at high solids load to achieve the desired product concentration (>50g/L) for economical product recovery. Microbial RNA isolation in the presence of high solids load is particularly challenging for bacteria, such as Clostridium thermocellum, that are tightly attached to the solid substrate. We tested several RNA isolation methods involving lysis methods including enzymatic (lysozyme) and/or mechanical (sonication and bead-beating with glass/zirconium/silica-carbide beads), in combination with Trizol® (or Trizol-Max®)/chloroform or RLT® buffer extraction, followed by RNeasy® cleanup of isolated RNA. Among these, cell lysis in Trizol® by pulsed sonication, followed by chloroform extraction and RNeasy® cleanup gave the best results with the highest yields and integrity of total RNA. The developed method can be used to study the physiology of C. thermocellum during biomass conversion processes operating with high loads of insoluble substrates and also expanded to isolate RNA from other microbes capable of utilizing biomass.
