Dog olfactory receptor gene expression profiling using samples derived from nasal epithelium brushing

Abstract

Dogs have an exquisite sense of olfaction. In many instances this ability has been utilized by humans for a wide range of important situations including detecting explosives and illegal drugs. It is accepted that some breeds have better senses of smell than others. Dogs can detect many volatile compounds at extremely low concentrations in air. To achieve such high levels of detection, the canine olfactory system is both complex and highly developed requiring a high density of olfactory receptors capable of detecting volatiles. Consequently the dog genome encodes a large number of olfactory receptor (OR) genes. However, it remains unclear as to what extent are all of these OR genes expressed on the cell surface. To facilitate such studies, a nasal brushing method was developed to recover dog nasal epithelial cell samples from which total RNA could be extracted and used to prepare high quality cDNA libraries. After capture by hybridization with an extensive set of oligonucleotides, the level of expression of each transcript was measured following next generation sequencing (NGS). The reproducibility of this sampling approach was checked by analyzing replicate samples from the same animal (up to 6 per each naris). The quality of the hybridization capture was also checked by analyzing two DNA libraries; this offered an advantage over RNA libraries by having an equal presence for each gene. Finally, we compared this brushing method performed on living dogs to a nasal epithelium biopsy approach applied to two euthanized terminally ill dogs, following consent from their owners.Comparison the expression levels of each transcript indicate that the ratios of expression between the highest and the least expressed OR in each sample are greater than 10,000 (paralog variation). Furthermore, it was clear that a number of OR genes are not expressed.The method developed and described here will allow researchers to further address whether variations observed in the OR transcriptome relate to dog 'life experiences' and whether any differences observed between samples are dog-specific or breed-specific.

Fig. 1

Schematic view of the sample localizations. from the Cane Corso and Golden Retriever euthanized dogs. Samples made by the brushing approach correspond to position 3. In addition, biopsy samples made from the Cane Corso and the Golden Retriever euthanized dogs correspond to positions 1 to 4 or 1 to 5 respectively

Fig. 2

Multiple samples clustering. Hierarchical clustering constructed with all OR FPKM log values, that correspond to the number of times each transcript was sequenced, which itself depend on their concentration in the libraries (Additional file 3). Most of the samples from the same animal are in the same cluster, the main exception are the two Belgian Shepherd samples, one of them being clustered with the Labrador Retriever samples, the second being alone. This overall good clustering indicates the good reproducibility of the sampling and of the analysis. The distribution of the two Belgian Shepperd samples in two different clusters suggests a pathological problem [30]

Fig. 3

Absence of correlation between the FPKM values and the number of expressed OR. This figure is made of four Tables (2a to 2D). Table 2a for each sample, the FPKM values of the most highly expressed OR (column 1). In column 2 are the number of OR expressed at a FPKM value ≥ to 1%. Column 3 indicates the number of OR expressed with an FPKM value ≥ to 1⁰/₀₀ and column 4 gives the total number of detected OR (FPKM ≥ 0.1). To check whether the FPKM values to which the highest expressed OR are detected impact the number of detected OR, we compared the number of detected OR (column 2, 3 or 4) to that of the highest FPKM values [1]. As shown in plots 2b, c and d there is no correlation between the highest FPKM values and the number of expressed OR genes indicating that the low number of detected OR is a reality and might be a characteristic of dogs