A gene expression signature of confinement in peripheral blood of red wolves (Canis rufus)
ABSTRACT: Background: The idea of using whole genome microarrays to detect peripheral blood biomarkers for physiological status a fairly new and unexplored method. Identifying biomarkers that can be linked to stress and immune response would be of great importance not only in animal management practices but in humans as well. The main objective of this research was to explore this concept using the North American Red Wolf (Canis rufus) which are exposed to a wide variety of environments from free ranging to confinement. Results: Transcription profiling of peripheral blood samples from 13 red wolf individuals in the Alligator River region of North Carolina revealed a strong signal of differentiation between confined and free-range animals. 482 out of 2,980 transcripts detected on the human Illumina Ref8 oligonucleotide bead arrays were found to differentiate these groups at a false discovery rate of 5 percent. Over-representation of genes in focal adhesion, insulin signaling, proteasomal, and tryptophan metabolism pathways suggests the activation of proinflammatory and stress responses in confined animals. Conclusions: Integration of immunological and physiological signals may leave a signature of lifestyle in the patterns of gene expression in the blood and suggest the possible development of biomarkers for disease and normal conditions. Keywords: Stress Response/Disease State Overall design: 14 whole blood samples were collect from captive and free ranging red wolves. Information pertaining to sex and current holding status was obtained for each individual. In total we had 6 captive wolves (2 females and 4 males), 7 free range wolves (2 females and 5 males) and 1 free range female coyote. Total RNA was extracted from whole blood samples stored in RNAlater at -20C. Total RNA was amplified and biotin labeled using Ambion's Illumina RNA Amplification Kit (#I1755). Each labeled sample was hybridized on 4 independent arrays, giving a total of 56 arrays (4/sample). Hybridization was done at 42C overnight. Arrays were washed and stained with strepavidin linked Cy3. Arrays were scanned on Illumina's BeadStation Scanner and raw intensity data was extracted using Illumina Beadstudio. Raw intensities were log base 2 transformed and then median center standardized in SAS using the proc stdize.
Project description:This study uses a custom made Nimblegen aCGH chip that targeted all segmental duplications in the canine genome to identify associated CNVs. A total of 23 hybridizations were performed in a panel of diverse dogs and a single wolf. This study focuses on the use a custom made Nimblegen aCGH chip to genotype 1,611 dog CNVs in 23 wolf-like canids (4 purebred dogs, one dingo, 15 gray wolves, one red wolf, one coyote and one golden jackal) to identify CNVs that may have arisen after domestication
Project description:Circulating miRNAs are an emerging class of biomarkers correlating their specific expression patterns to disease states. A considerable proportion of hematopoietically-derived miRNAs are present in plasma with the ability to confound the signature of true circulating miRNA species. We use microarray analysis to catalogue a list of 313 haemotopoetic miRNAs and analyze expression profiles of cell-free miRNAs in individual plasma fractions after calibrating for cellular miRNA signals. Comprehensive global maps of bona fide circulating miRNA species are presented, and inter-individual variability and gender-specific expression is explored in populations of healthy individuals. Healthy Caucasian individuals were used to segregate blood into 8 subfractions: white blood cells (WBC)/buffy coat, leukocytes, red blood cells (red blood cells (RBC)), cloudy supernatant (CS), supernatant 1 (S1), supernatant 2 (S2), pellet 1 (P1) and pellet 2 (P2) through differential centrifugation to understand the proportion of cellular miRNAs in each category.
Project description:PBMC were exposed to Cell Free Nucleic Acids (CFNA) extracted from blood products eligible for transfusion: Red Blood Cell units (RBC), Fresh Frozen Plasma units (FFP). PBMC alone exposed to the same extract from PBS were used as a control (PBMC+control). PBMC alone were also used as control. The used positive control was PBMC exposed to Lipopolysaccharide (LPS) Overall design: exposition of PBMC to CFNA, RNA extarction and gene expression array (24h) PFC = FFP = Fresh Frozen Plasma units CE = RBC = Red Blood Cell units CP = PC = Platelet Concentrates
Project description:Background: Aquaculture of the black tiger prawn Penaeus monodon remains severely constrained by an almost total dependence on wild-caught broodstock. Reliance on wild-caught broodstock stems, for the most part, from reduced reproductive potential of captive-reared females. Reproductive performance of captive-reared females is usually characterised by longer latency period, lower egg production, egg hatch rates and post-larval survivorship compared with their wild-caught counterparts. Improved understanding of the cellular and associated molecular events occurring during peneaid ovarian maturation could therefore be fundamental to improving reproductive success of captive-reared animals. Methodology/Principle Findings: In support of other studies, our histological analyses of developing oocytes revealed differences between wild-caught and captive-reared P. monodon, including reduced lipid accumulation in oocytes of captive-reared animals. We have employed oligonucleotide microarray analysis to compare expression profiles of genes involved in ovarian maturation among wild-caught and captive-reared animals. Custom oligonucleotide microarrays were constructed and screened with transcripts derived from the ovary, cephalothorax and eyestalk from animals of all ovarian maturation stages. Ovarian maturation-related differential expression patterns were observed for 111 transcripts, with 53 transcripts displaying differential expression between wild-caught and captive-reared animals. Notably transcripts encoding vitellogenin - the major egg yolk protein precursor, and a lipid storage droplet protein (which we named pmLSD) which is involved in lipid accumulation, were found to be more highly expressed in wild-caught animals. pmLSD transcripts localise to pre-vitellogenic oocytes of wild-caught animals and the pmLSD protein is exclusively localised to the surface of lipid droplets of oocytes at vitellogenic and cortical rod stages. We have employed oligonucleotide microarray analysis to compare expression profiles of genes involved in ovarian maturation among wild-caught and captive-reared animals. Target preparation and microarray hybridisation. Ovarian RNA samples from nine wild-caught animals representing six ovarian maturation stages (P, 2, 24, V, R, E) were used in microarray hybridisations. Similarly, RNA samples from three captive-reared animals representing four maturation stages (P, 24, V, E) were used in microarray hybridisations. For wild-caught animals, samples from each ovarian maturation stage were pooled into groups of four and five, enabling two hybridisations. For captive-reared animals, samples from each ovarian maturation stage from all three animals were pooled enabling one hybridisation for each stage. Importantly, as the four stages for captive-reared animals were (1) pre-ablation pre-vitellogenic, (2) post-ablation pre-vitellogenic, (3) post-ablation vitellogenic, (4) post-ablation vitellogenic with cortical rods, this arrangement allowed for 2 samples of captive-reared pre-vitellogenic and 2 samples of captive-reared vitellogenic, thereby enabling t-tests between samples, while also allowing analysis across the whole 4 stages via cluster analysis. All hybridisations were single channel hybridisations conducted using equal amounts of RNA pooled from each individual.
Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC Overall design: There are 10 samples for each of PBMC, PAX and PAX globin-reduced, where 5 samples come from sickle-cell patients and 5 from healthy controls.
Project description:Four captive bottlenose dolpins housed in Kona, Hawaii, were serially sampled at approximately monthly intervals over the course of one year, in order to establish baseline information on the content and variation of the dolphin blood transcriptome. Overall design: Illumina based RNA-seq analyses were carried out on globin-depleted RNA from blood using the Ensembl dolphin genome as a guide. The data set was interrogated for transcripts exhibiting circa-annual rhymthicity, differential expression by season or sex, or correlation to hematological parameters
Project description:The mammalian oviduct is a dynamic organ and the venue of important events leading to the establishment of pregnancy. Oviductal epithelial cells are involved in direct contact and specific interactions with different self and non-self entities (oocyte, sperm, etc.).The aim of this study was to determine if the oviduct is able to distinguish between different types of non-self-entities. We used an in vitro model to determine genes altered in oviductal epithelial cell (OPEC) in response to the presence of spermatozoa, mammalian somatic cells (red blood cell) or bacteria. We hypothesised that alteration of OPEC expression profile in response to boar spermatozoa (friendly non-self entity), red blood cells (non-pathogenic, non-self entity) and bacteria (pathogenic non-self entity) is indicative of recognition of self and non-self entities by these cells as well as recognition of pathogenic from non-pathogenic non-self entities. Oviductal epithelial cells (OPEC) were co-incubated in the presence of (i) bacteria (1 x 10˄8 bacteria), (ii) sperm (1 X 10 ˄ 6 spermatozoa/ml) or (iii) red blood cell (5 x 10 ˄ 6 red blood cell/ml) for 24 hours in HAM-12 media. Three biological replicates were performed for each group and a total of 12 Affymetrix Porcine arrays were used to identify the expression profiles of OPEC alone (3 arrays) or in the presence of (i) bacteria (3 arrays), (ii) sperm (3 arrays) or (iii) red blood cells (3 arrays).
Project description:RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133Plus 2.0 arrays were hybridized with probe from total RNA isolated from blood sampled from 6 umbilical cords and 6 healthy adult humans. Overall design: Peripheral blood from 6 normal adults and 6 human umbilical cords were purified to obtain packed red blood cells and their purity was assessed by CELL-DYN4000. The contaminated WBC count was less than 1 out of 1 million cells. Total RNA from purified reticulocytes included in the purified red blood cells was extracted, labeled, and hybridized onto Affymetrix HG-U133Plus 2.0 arrays.