Feline tooth transcriptome changes involvement in feline tooth resorption (TR)

Advanced next generation sequencing approaches have started to reveal the cellular and molecular complexity of microenvironment in many tissues. As clinical archived dental samples derived from humans and animals are valuable resources in dental research, there is a necessity to maximise utility of such samples for research purposes. However, it is challenging to obtain high quality and good yield of RNA especially in mineralised tissues such as bone and teeth. We compared two methods and developed an optimised method of RNA extraction from feline teeth collected in a clinical setting and at post mortem. Tooth samples were homogenised in phenol-guanidinium solution while maintaining near-freezing temperatures and followed by solid-phase nucleic acid extraction utilising a commercially available kit. This method produced a good yield of RNA ranging from 2.0 to 12 µg of total RNA from teeth with a minimum wet weight of 100 mg. We report that this optimised protocol improved the quality of RNA based on RNA integrity numbers equivalent (RINe) from an average of 3.6 to 5.6. These RINe values were lower than those of RNA extracted from soft tissues but this limitation has been previously reported for mineralised tissues. There was no correlation found between RNA purity parameters measured by the A260:280 or A230:260 ratios and the degree of RNA degradation defined as RINe. This implies that these RNA purity indicators cannot be reliably used as parameters of RNA integrity and quality. The stability of reference gene expression varied depending on the choice of reference genes rather than the degree of RNA degradation. Furthermore, we investigated the effect of quantity and quality of RNA from feline teeth on RNA-seq results. Thirteen RNA-seq data showed similar duplication rates and high mapping rates (94 to 95%) against the feline genome regardless of RINe value. However one low yield sample with a high RINe score showed a high duplication rate and was clearly represented as an outlier on the RNA-seq multidimensional scaling plot. We concluded that the overall yield of the RNA for input into RNA-seq was more important than quality of RNA for RNA-seq quality control. These results will hopefully guide others who wish to perform RNA extractions from mineralised tissues, especially when the tissues have to be collected in a clinical setting with the restraints that impose on a study.