Roberts, S. and Snyder, L. (2012) Comparison of RNA extraction methods for effective isolation of bacterial RNA from neisserial cultures. In: XVIIIth International Pathogenic Neisseria Conference (IPNC); 09 - 14 Sep 2012, Würzburg, Germany. (Unpublished)

Abstract

RNA is an important macromolecule isolated in many areas of research to look at biological reactions, gene expression, cellular signals, processing, and more. However, in order to look into specific areas of interest, purification is required before any type of downstream transcriptomics work, such as sequencing, microarrays, and quantitative RT-PCR, to ensure reliability and success. In this research, three different RNA extraction methods were compared using both widely available reagents as well as commercial kits. Each method was compared to establish how efficient and effective it was in extracting pure RNA using the Gram-negative bacterial species, Neisseria gonorrhoeae strain NCCP11945. RNA was extracted from cultures using the Ambion® RiboPure™ Kit (Life Technologies™), the RNeasy Kit (QIAGEN®) and a previously reported combined method using TRIzol® Reagent (Life Technologies™) followed by the RNeasy Kit. Evaluation of RNA quantity and purity was carried out using the Nanovue® spectrophotometer and quality and integrity was established using the Agilent 2100 Bioanalyzer and agarose gels. There was little success with the Ambion® RiboPure™ Kit for extraction of N. gonorrhoeae RNA. Although the TRIzol/ RNeasy-based extraction was successful at producing high yields it used hazardous chemicals and required further purification methods. It was concluded that the RNeasy kit used on its own was proven to be the best method of extraction in terms of RNA yield, quality, reproducibility, and convenience.

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