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Leukocyte count affects expression of reference genes in canine whole blood samples.

ABSTRACT: BACKGROUND: The dog is frequently used as a model for hematologic human diseases. In this study the suitability of nine potential reference genes for quantitative RT-PCR studies in canine whole blood was investigated. FINDINGS: The expression of these genes was measured in whole blood samples of 263 individual dogs, representing 73 different breeds and a group of 40 mixed breed dogs, categorized into healthy dogs and dogs with internal and hematological diseases, and dogs that underwent a surgical procedure. GeNorm analysis revealed that a combination of 5 to 6 of the most stably expressed genes constituted a stable normalizing factor. Evaluation of the expression revealed different ranking of reference genes in Normfinder and GeNorm. The disease category and the white blood cell count significantly affected reference gene expression. CONCLUSIONS: The discrepancy between the ranking of reference genes in this study by Normfinder and Genorm can be explained by differences between the experimental groups such as "disease category" and "WBC count". This stresses the importance of assessing the expression stability of potential reference genes for gene experiments in canine whole blood anew for each specific experimental condition.

SUBMITTER: Piek CJ

PROVIDER: S-EPMC3045956 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Leukocyte count affects expression of reference genes in canine whole blood samples.

Piek Christine J CJ Brinkhof Bas B Rothuizen Jan J Dekker Aldo A Penning Louis C LC

BMC research notes 20110209

<h4>Background</h4>The dog is frequently used as a model for hematologic human diseases. In this study the suitability of nine potential reference genes for quantitative RT-PCR studies in canine whole blood was investigated.<h4>Findings</h4>The expression of these genes was measured in whole blood samples of 263 individual dogs, representing 73 different breeds and a group of 40 mixed breed dogs, categorized into healthy dogs and dogs with internal and hematological diseases, and dogs that under ...[more]

PMID: 21303565

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Project description:BackgroundPreanalytic factors such as time and temperature can have significant effects on laboratory test results. For example, ammonium concentration will increase 31% in blood samples stored at room temperature for 30 min before centrifugation. To reduce preanalytic error, blood samples may be placed in precooled tubes and chilled on ice or in ice water baths; however, the effectiveness of these modalities in cooling blood samples has not been formally evaluated. The purpose of this study was to evaluate the effectiveness of various cooling modalities on reducing temperature of EDTA whole blood samples.MethodsPooled samples of canine EDTA whole blood were divided into two aliquots. Saline was added to one aliquot to produce a packed cell volume (PCV) of 40% and to the second aliquot to produce a PCV of 20% (simulated anemia). Thirty samples from each aliquot were warmed to 37.7 °C and cooled in 2 ml allotments under one of three conditions: in ice, in ice after transfer to a precooled tube, or in an ice water bath. Temperature of each sample was recorded at one minute intervals for 15 min.ResultsWithin treatment conditions, sample PCV had no significant effect on cooling. Cooling in ice water was significantly faster than cooling in ice only or transferring the sample to a precooled tube and cooling it on ice. Mean temperature of samples cooled in ice water was significantly lower at 15 min than mean temperatures of those cooled in ice, whether or not the tube was precooled. By 4 min, samples cooled in an ice water bath had reached mean temperatures less than 4 °C (refrigeration temperature), while samples cooled in other conditions remained above 4.0 °C for at least 11 min. For samples with a PCV of 40%, precooling the tube had no significant effect on rate of cooling on ice. For samples with a PCV of 20%, transfer to a precooled tube resulted in a significantly faster rate of cooling than direct placement of the warmed tube onto ice.DiscussionCanine EDTA whole blood samples cool most rapidly and to a greater degree when placed in an ice-water bath rather than in ice. Samples stored on ice water can rapidly drop below normal refrigeration temperatures; this should be taken into consideration when using this cooling modality.

Project description:BACKGROUND: The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Real-time reverse transcription PCR and a mathematical algorithm were used to establish which reference genes were most stably expressed in normal and diseased canine articular tissues and two canine cell lines stimulated with lipolysaccaride (LPS). RESULTS: The optimal reference genes for comparing gene expression data between normal and diseased infrapatella fat pad were RPL13A and YWHAZ (M = 0.56). The ideal reference genes for comparing normal and osteoarthritic (OA) cartilage were RPL13A and SDHA (M = 0.57). The best reference genes for comparing normal and ruptured canine cranial cruciate ligament were B2M and TBP (M = 0.59). The best reference genes for normalising gene expression data from normal and LPS stimulated cell lines were SDHA and YWHAZ (K6) or SDHA and HMBS (DH82), which had expression stability (M) values of 0.05 (K6) and 0.07 (DH82) respectively. The number of reference genes required to reduce pairwise variation (V) to <0.20 was 4 for cell lines, 5 for cartilage, 7 for cranial cruciate ligament and 8 for fat tissue. Reference gene stability was not related to the level of gene expression. CONCLUSION: The reference genes demonstrating the most stable expression within each different canine articular tissue were identified, but no single reference gene was identified as having stable expression in all different tissue types. This study underlines the necessity to select reference genes on the basis of tissue and disease specific expression profile evaluation and highlights the requirement for the identification of new reference genes with greater expression stability for use in canine articular tissue gene expression studies.

Project description:In early lactation, an energy deficit leading to a negative energy balance (NEB) is associated with increased susceptibility to disease and has been shown to be an important factor during transition in dairy cows. L-carnitine as a key factor in the mitochondrial transport of fatty acids and subsequently for β-oxidation and energy release is known to modulate mitochondrial biogenesis and thus influence metabolism and immune system. In the current study, we characterized hematological changes around parturition and investigated the potential effects of dietary L-carnitine supplementation on immune cell functions. For this approach, dairy cows were assigned either to a control (CON, n = 30) or an L-carnitine group [CAR, n = 29, 25 g rumen-protected L-carnitine per cow and day (d)]. Blood samples were taken from d 42 ante partum (ap) until d 110 post-partum (pp), with special focus and frequent sampling from 0.5 to72 h post-calving to clarify the impact of L-carnitine supplementation on leukocyte count, formation of reactive oxygen species (ROS) in polymorphonuclear cells (PMN) and peripheral mononuclear cells (PBMC) and their phagocytosis activity. Blood cortisol concentration and the capacity of PBMC proliferation was also investigated. All populations of leukocytes were changed during the peripartal period, especially granulocytes showed a characteristic increase up to 4 h pp. L-carnitine supplementation resulted in increased levels of eosinophils which was particularly pronounced one day before to 4 h pp, indicating a possible enhanced support for tissue repair and recovery. Non-supplemented cows showed a higher phagocytic activity in PBMC as well as a higher phagocytic capacity of PMN during the most demanding period around parturition, which may relate to a decrease in plasma levels of non-esterified fatty acids reported previously. L-carnitine, on the other hand, led to an increased efficiency to form ROS in stimulated PMN. Finally, a short period around calving proved to be a sensitive period in which L-carnitine administration was effective.

Project description:BackgroundPreterm birth is the leading cause of neonatal morbidity and mortality, but research efforts in neonatology are complicated due to the unavailability of large volume blood samples. Whole blood assays can be used to overcome this problem by performing both functional and gene expression studies using small amounts of blood. Gene expression studies using RT-qPCR estimate mRNA-levels of target genes normalized to reference genes. The goal of this study was to identify and validate stable reference genes applicable to cord blood samples obtained from developing neonates of different gestational age groups as well as to adult peripheral blood samples. Eight reference gene candidates (ACTB, B2M, GAPDH, GUSB, HPRT, PPIB, RPLP0, RPL13) were analyzed using the three published software algorithms Bestkeeper, GeNorm and NormFinder.ResultsA normalization factor consisting of ACTB and PPIB allows for comparative expression analyses of neonatal samples from different gestational age groups. Normalization factors consisting of GAPDH and PPIB or ACTB and GAPDH are suitable when samples from preterm and full-term neonates and adults are compared. However, all candidate reference genes except RPLP0 exhibited significant intergroup gene expression variance and a higher gene expression towards an older age which resulted in a small but statistically significant systematic bias. Systematic analysis of RNA-seq data revealed new reference gene candidates with potentially superior stability.ConclusionsThe current study identified suitable normalization factors and proposed the use of the additional single gene RPLP0 to avoid systematic bias. This combination will enable comparative analyses not only between neonates of different gestational ages, but also between neonates and adults, as it facilitates more detailed investigations of developmental gene expression changes. The use of software algorithms did not prevent unintended systematic bias. This generally highlights the need for careful validation of such results to prevent false interpretation of potential age-dependent changes in gene expression. To identify the most stable reference genes in the future, RNA-seq based global approaches are recommended.

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Leukocyte count affects expression of reference genes in canine whole blood samples.

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Leukocyte count affects expression of reference genes in canine whole blood samples.

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