The researchers used total RNA from well-characterized microbial culture samples individually, as well as a mixed pool, for benchmarking rRNA removal methods:
- Epicentre Ribo-Zero™ Meta-Bacteria Kit (solution-based hybridization capture);
- NuGEN Ovation Prokaryotic RNA-Seq System (NuGEN) (proprietary set of "not so random" primers to avoid rRNA as template during first and second strand cDNA synthesis);
- Duplex-Specific Nuclease (DSN; degradation of fast re-annealing abundant cDNAs to preferentially deplete ribosomal cDNA);
- Life Technologies MicrobEXPRESS Kit (solution-based hybridization capture); and
- Epicentre Terminator Exonuclease (degrades RNAs with a 5'-monophosphate group).
The results demonstrated the superiority of the Ribo-Zero Kit in reducing rRNA in the microbial samples. In artificially degraded samples and in the clinical samples, the Ribo-Zero Kit performed well, while some of the other methods performed poorly or failed completely at removing rRNA. The authors further note the potential for simple automation of the Ribo-Zero procedure, and that the Ribo-Zero process can be applied to profile gene expression in both simple and complex, naturally occurring bacterial communities.
Giannoukos, G. et al. (2012). Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes Genome Biology, 13 (3) DOI: 10.1186/gb-2012-13-3-r23