Optimization of RNA Extraction for Oral Metatranscriptome Studies

Objectives:
Characterization of the oral metatranscriptome through high throughput RNA sequencing (RNA-seq) is an indispensable tool to understand the functionality of the oral microbiome. However, RNA extraction from polymicrobial communities poses certain challenges such as the presence of species with different cell wall characteristics and low quantities of starting material. The aim of this study was to optimize RNA isolation from oral microorganisms for the purposes of RNA seq.
Methods: Different cell lysis methods, commercially available kits for RNA extraction and DNA removal strategies were tested on cultures of two common oral species, the Gram-positive Actinomyces oris and the Gram-negative Porphyromonas gingivalis. Microorganisms were grown under appropriate conditions, diluted to concentrations equivalent to 10^7 or 10^9 cell mL^-1 and frozen in RNA Cell protect (Qiagen). Cell lysis was performed via membrane disruption with 0.1 mm silica beads for 40 or 80 seconds in a bead beater or by 10 min vortexing. Two RNA isolation kits, the PowerMicrobiome RNA Isolation kit (MoBio) and the RNeasy Isolation kit (Qiagen), and two DNA removal methods, the Turbo-free DNAse (Life technologies) and in-column DNAse digestion (Qiagen), were tested. RNA quantity was measured using Qubit RNA HS Assay Kit and quality was measured using the Agilent BioAnalyzer, which assigns a RNA Integrity Number (RIN) to each sample. A RIN of 7 was set as the minimum quality measure.
Results: Bead vortexing in combination with the Qiagen RNeasy kit and the Qiagen in-column DNAse digestion provided the best combination of high yield and good quality RNA for both species.
Conclusions: By using two microorganisms with different cell wall characteristics, we have optimized an RNA extraction protocol yielding RNA of sufficient quantity and quality for downstream RNA-seq applications.