Subjects in the current study
Brain and temporal muscle samples from five donated animals were included in the validation of the sampling protocol. All used samples were identified by the animal ID numbers and additional sample ID numbers (see Fig 2). The animals included in the validation study were the following: ID:170529–1: male Labrador retriever, 13 years old; ID:170713–1: male Labrador retriever, 14 years old; ID:170905–1 female mongrel, 13 years old; ID:171102–1 female Beagle, 3 years old; ID:171102–2 female Beagle, 3 years old. Brain samples from one donated animal were used to assess the effect of RNAlater fixation on mRNA integrity: a male Labrador retriever, 13 years old (ID:180601–1).
Samples of the Canine Brain and Tissue Bank (CBTB) originated from voluntary donations of owners whose dogs were euthanized due to medical reasons. The possibility of donations to the CBTB has been advertised through various social forums and media. Prior to the actual donation, both the owners and the veterinarians, who performed the euthanasia, had to fill in a “statement of donation” document, which contained a “statement of consent” to be filled by the veterinarian. Without that statement (which included statements of medical necessity and suitability) the donations were not accepted. Other criteria for accepting a donation included valid anti-rabies vaccination of the animal.
If the veterinarian / owner gave consent (by filling the appropriate field of the “statement of consent” document) for collecting and storing data about the animal, the medical records and any other known record (e.g. results of previous behaviour tests, questionnaires) were also obtained.
Euthanasia of the animals were performed at veterinary clinics and the cadavers were subsequently transported to the facility of the CBTB where the sampling was performed according to the predefined protocols. We set a 4 hours post-mortem delay as limit for sample fixation for molecular investigations, based on previous findings  and considering the expected maximal transportation times of donations from the clinics to the facility. The post-mortem interval between death of the animal and immersion of a tissue piece into RNAlater were recorded for every obtained sample (1 tissue piece = 1 sample).
For molecular research, brain samples were taken from the frontal cortex, cerebellum and brain stem, and muscle samples were collected from the temporal muscle, bilaterally (a total of eight samples / animal).
To obtain molecular grade tissue samples, 80 - 120 mg pieces from each site (e.g. a piece of cortex from the left side of the brain) were put into 1 ml RNAlater. After immersion in RNAlater, the samples were incubated overnight at 4°C before being frozen at –20°C. For long term storage, the RNAlater fixed tissue samples were transferred to –80°C following removal of the supernatant.
After obtaining the tissue pieces intended for molecular investigations, the two hemispheres of the brain were separated and stored under different conditions. The left hemisphere was put into 4% buffered formaldehyde and stored at 4°C. The right hemisphere was stored in an ULT freezer at –80°C degrees. In the current study, these samples (the formaldehyde fixed or frozen hemispheres) were not included. In the case of donations that exceeded the 4 hours time limit, samples were not included in the molecular research, and these brains (both hemispheres) were fixed in formaldehyde for histological purposes.
We performed two experiments to assess the quality of the obtained samples and the effect of the applied fixation / storage method.
In the first experiment, we assessed the stability of the three chosen reference genes in the three brain regions and muscle of five individuals using the samples collected and stored by our standard procedure (RNAlater fixation).
In the second experiment, we compared the Ct values of paired RNA samples derived from one animal: one half of the same tissue sample was put into RNAlater after removal from the body, while the other half was immediately put into TRIzol for RNA isolation. Therefore, we could assess whether RNAlater stabilization had an effect on the detected Ct values of the three housekeeping genes. Only brain samples (frontal cortex, cerebellum and brain stem, bilaterally) were used for this analysis.
Total cellular RNAs were isolated using TRIzol (Thermo Fisher Scientific) and following the manufacturer’s protocol. For the isolation, smaller portions (30–50 mg) were cut from the RNAlater fixed tissue pieces and were rinsed in 1 ml sterile PBS before they being immersed in 500 ml TRIzol. Homogenization of the tissue pieces was done by an Ultra-Turrax homogenizer (Ika).
Quality of the isolates were checked by agarose gel electrophoresis, and the concentrations were measured by a NanoDrop device (Thermo Fisher Scientific). The isolates used for RT-qPCR all had a very similar S18/S28 ratio based on the gel electrophoresis results.
Isolated RNA samples were stored at –80°C for long term.
- cDNA synthesis and RT-qPCR
1000 ng of each total RNA was reverse-transcribed into cDNA using the Maxima RevertAid cDNA Synthesis Kit (Thermo Fisher Scientific) and following the manufacturer’s protocol, with random hexamer primers. Prior to downstream applications, the cDNA samples were diluted tenfold by nuclease-free water and kept at –20°C. For long-term storage, the cDNA samples were placed at –80°C.
Quantitative Real-Time PCR reactions were performed on a StepOne Plus Instrument (Thermo Fisher Scientific) using commercial TaqMan assays and the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific). The following TaqMan assays were used to target three genes, which were previously validated as reference genes for dog brain samples : Cf04419463_gH for GAPDH; Cf02690456_g1 for HPRT1; Cf02694648_m1 for HMBS. Each reaction was performed in triplicate on a 96 well plate. Both non-template controls and negative controls were applied during the experiment. Negative controls contained non-transcribed RNA in the same dilution as in reaction mixes containing transcribed cDNA.
All reference genes were detected in all tissue samples tested. The efficiencies of the assays were determined by a five step dilution curve and were between 92% and 93% for all assays. Because of the similar efficiency values, we assumed that the absolute expression levels of the genes were comparable in this setting.
Importantly, in some cases inter-assay comparisons showed more than 2 cycle threshold (Ct) differences between replicated measurements, while intra-assay relative values between different samples were more consistent, as shown by the example of HPRT1 (Fig. 1). The inter-assay average difference between same samples was found to be 3.03, while the inter-assay difference between relative Ct values (Ct of each sample compared to the average of each plate) was only 0.44. This was in accordance with the literature  regarding the higher consistency of relative Ct values.
As the capacity of plates used for our study allowed for a maximum of 96 reactions simultaneously, we assigned samples to provide the best grouping of parameters within the same plate in order to minimize the noise of inter-assay comparisons of absolute Ct values of the reference genes. For example, same tissue types were measured for all individuals on the same plate for each gene to minimize inter-assay noise of inter-individual differences.
Since the binding site of the assay primers can also modify results, because the 3’ ends of mRNAs are more prone to degradation , we recorded the position of primer recognition sites on the mRNAs. In our study, the assays for HPRT1 and HMBS recognized exons boundaries close to the 3’ end, while the GAPDH assay was positioned in the middle of the reference mRNA sequence.
- Software and statistical analysis
The absolute Ct means (derived from the three technical parallels run on a plate) belonging to different individuals were analysed in each tissue type to assess the stability of the reference genes. In the brain, comparisons were done in each investigated region (frontal cortex, cerebellum, brain stem) and for the whole brain altogether (regions grouped together).
We used MS Excel to determine standard deviations (SD), and we used the NormFinder  and BestKeeper  applications for detailed analyses of the stability of the reference genes.
We used the IBM SPSS Statistics software version 25 for statistical analysis. We used one-way ANOVA for pairwise comparisons of the absolute Cts belonging to each gene in all settings (3 brain region separately and together and muscle separately). As we analysed a total of five individuals, N was 5 for each gene in each region, except for the “whole brain” setting, where N was 15. Based on the number of genes, K was 3.