Project description:Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and -80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P < 0.001), storage temperature (F-value=1.77, DF = 3, P = 0.037), and duration of sample storage (F-value = 3.68, DF = 3, P < 0.001). Changes in bacterial composition were observed in samples stored in -80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF = 8.57, P < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

Project description:Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq proved more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas.

Project description:Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq proved more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas. RNA was extracted from 10 lymphoma fine needle aspirates attained from companion canines. 4 normal lymph node samples were obtained from a Beagle breeding colony at Pfizer, including two samples that were taken from the same dog but different lymph nodes. This Series represents the Affymetrix gene expression only, not RNA-Seq referenced above. RNA-Seq data have been submitted to SRA as SRA059558.

Project description:BackgroundGreenland sled dogs (GSD) are a unique, genetically isolated population of dogs living under exceptional environmental conditions. Metabolism, and thereby thyroid hormones are affected by multiple factors. Among other activity, energy balance and environmental conditions are important. A breed-specific reference interval (RI) can be useful for diagnostics of potential thyroid-related pathologies. The aim of this study was to establish RIs of the thyroid hormones thyroxin (T4), free thyroxin (fT4), and thyroid stimulating hormone (TSH) in GSD. In addition to evaluate the effect of sex, age, season, management, and body condition score (BCS) in GSD. Physical exams and cephalic venous blood sampling were performed in the period of 2018-2019 from 265 GSD managed either privately or by the Danish navy. Serum biochemical analyses, including C-reactive protein, were performed and RIs were determined for TSH, T4 and fT4 in only healthy dogs. The RIs were determined using American Society for Veterinary Clinical Pathology guidelines and the effect of varying factors were evaluated by linear regression and further tested by Mann-Whitney test.Results144 GSD were included in the reference group resulting in RIs: T4: 6.44-48.65 nmol/L; fT4: 3.91-18.51 pmol/L; and TSH: 0.04-0.55 ng/mL. Female GSD had significantly higher concentrations of T4 (P = 0.039) and fT4 (P = 0.015) compared to males; a positive correlation between TSH and aging was found; T4 concentrations were significantly higher (P = 0.003) during summer; and TSH concentrations were lower in GSD managed by the navy (P < 0.0001). BCS was higher (P < 0.0001) in Sirius GSD compared to civilian GSD, and BCS was positively correlated with T4 and negatively correlated with TSH.ConclusionsReference intervals for T4, fT4 and TSH in GSD were established. The RI for T4 and fT4 was lower compared to other breeds. In addition, sex, age, season, management and BCS demonstrated variable effects on thyroid hormones. Our results can be used as a foundation for improving management and further research of GSD.

Dataset Information

Comprehensive annotation and evolutionary insights into the canine (Canis lupus familiaris) antigen receptor loci.

Publications

Comprehensive annotation and evolutionary insights into the canine (Canis lupus familiaris) antigen receptor loci.

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