Comparison of RNA extraction methods for effective isolation of bacterial RNA from neisserial cultures

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