An efficient method for extracting next-generation sequencing quality RNA from liver tissue of recalcitrant animal species.

ABSTRACT

The next-generation RNA sequencing technologies expedite the discovery of a large number of novel transcripts and genes associated with various pathophysiological conditions. These technologies involve poly(A) enrichment, which in turn requires micrograms of high-quality total RNA. Unfortunately, the available RNA isolation approaches produce poor quality total RNA from difficult-to-isolate animal tissues, such as the liver with high glycogen content. Moreover, the extraction efficiencies of these approaches vary significantly depending on the animal species. To address this challenge, we optimized a three-step protocol for the extraction of high-yield and high-quality total RNA from the liver tissue (LT). The procedure effectively resolved the problem of glycogen coprecipitation by its stepwise removal. No signs of RNA degradation on gel electrophoresis analysis and RNA integrity number values ≥8.5 indicated that the extracted RNA is suitable for downstream processing, such as poly(A) enrichment and transcriptome profiling. To demonstrate the robustness of the novel protocol, a comparison was made with other currently available RNA extraction approaches from diverse resources. Whereas other protocols yielded partially degraded bands with either decreased or reversed ribosomal RNA (rRNA) ratio, our protocol yielded intact rRNA with a ratio of 2.0 ± 0.1. This optimized protocol was also successfully followed for other animal tissues, such as the bone and muscles. In conclusion, the study has described a highly efficient method for the next-generation sequencing quality RNA isolation from LT across a broad range of animal species, with extended applicability to other difficult-to-isolate tissues.