Abstract
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.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Teeth consist of connective tissues and highly specialised cells that produce a unique extracellular matrix composed of organic matrix proteins and inorganic minerals
Selection of high quality and quantity RNA is important for downstream gene expression studies using generation sequencing
tooth resorption (TR) affected tissues undergo apoptotic processes and stimulate inflammation which can lead to extensive loss of tissues (Dupont and Debowes 2002; Booij-vrieling et al 2010)
Summary
Teeth consist of connective tissues and highly specialised cells that produce a unique extracellular matrix composed of organic matrix proteins and inorganic minerals. Alveolar bone and deciduous teeth resorption during tooth eruption and shedding is a complex process that is tightly regulated at the molecular level (Ten Cate and Nanci 2003; Bei 2009). In permanent teeth, mineralised tissues such as enamel, cementum and dentine do not turn over or have limited regeneration capacities. The hard tissues of the tooth can be resorbed under pathologic conditions such as inflammation, mechanical trauma or idiopathic tooth resorption (TR) (Darcey and Qualtrough 2013). The exact mechanisms behind the pathological resorption observed in idiopathic TR are still unclear.
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