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Optimised isolation method for RNA extraction suitable for RNA sequencing from feline teeth collected in a clinical setting and at post mortem
- Lee, S., Trivedi, U., Johnson, C., Farquharson, C., Bergkvist, G. T.
- Veterinary research communications 2019 v.43 no.1 pp. 17-27
- RNA, analytical kits, cats, genes, high-throughput nucleotide sequencing, isolation techniques, mineralization, multidimensional scaling, quality control, quantitative polymerase chain reaction, teeth, temperature, tissues
- Advanced next generation sequencing approaches have started to reveal the cellular and molecular complexity of the microenvironment in many tissues. It is challenging to obtain high quality RNA from mineralised tissues. We developed an optimised method of RNA extraction from feline teeth collected in a clinical setting and at post mortem. Teeth were homogenised in phenol-guanidinium solution at near-freezing temperatures and followed by solid-phase nucleic acid extraction utilising a commercially available kit. This method produced good RNA yields and improved RNA quality based on RNA integrity numbers equivalent (RINᵉ) from an average of 3.6 to 5.6. No correlation was found between RNA purity parameters measured by A₂₆₀:₂₈₀ or A₂₃₀:₂₆₀ ratios and degree of RNA degradation. This implies that RNA purity indicators cannot be reliably used as parameters of RNA integrity. Two reference genes (GAPDH, RPS19) showed significant changes in expression levels by qPCR at low and moderate RINᵉ values, while RPL17 was stable at all RINᵉ values tested. Furthermore, we investigated the effect of quantity and quality of RNA on the quality of the resultant RNA sequencing (RNA-Seq) data. Thirteen RNA-seq data showed similar duplication and mapping rates (94 to 95%) against the feline genome regardless of RINᵉ values. However one low yield sample with a high RINᵉ value showed a high duplication rate and it was an outlier on the RNA-seq multidimensional scaling plot. We conclude that the overall yield of RNA was more important than quality of RNA for RNA-seq quality control. These results will guide researchers who wish to perform RNA extractions from mineralised tissues, especially if collecting in a clinical setting with the recognised restraints that this imposes.