Isolation of High-Quality RNA from Pichia pastoris.

Abstract

Analysis of RNA structuromes provides new insights into cellular processes, enabling systems biology and biotechnology researchers to calculate promoter and terminator strengths and to directly observe how differing circuit states impact host gene expression and the burdens imposed by the circuits. Such analysis, however, is crucially dependent on the availability of highly pure, intact RNA isolated from fresh or frozen cell cultures. RNA extraction from the yeast Pichia pastorisrequires specific pretreatment steps to ensure the reproducibility of downstream applications, but current methods and extraction kits are generally adapted for the conventional yeast Saccharomyces cerevisiae, which has a different cell wall composition. We therefore set out to compare the efficacy of two different RNA isolation methods when applied to P. pastoris: (i) phenol/chloroform extraction and (ii) silica spin-column absorption. We compared the yield, integrity, and purity of the resulting isolated RNA from the two methods (using two different types of commercial columns for silica spin-column absorption) and further optimized them through variations in the pretreatment steps. We also assessed two different methods of cell lysis: enzyme catalytic disruption using lyticase and mechanical disruption using acid-washed glass-beads in a TissueLyser. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: RNA isolation with phenol/chloroform extraction: monophasic lysis reagent Alternate Protocol 1: RNA isolation with silica-spin column absorption: High Pure RNA Isolation Kit (Roche Life Science) Alternate Protocol 2: RNA isolation with silica-spin column absorption: RNeasy Mini Kit (Qiagen).