Validation of internal control genes for gene expression analysis in bovine polymorphonuclear leukocytes

Analysis of gene expression is becoming more important in all areas of biological research to evaluate gene expression during physiological and pathological conditions (e.g., mastitis), not the least in the field of animal research. Presently, real-time gene expression analysis is considered to be the method of choice for accurate and sensitive quantification of mRNA transcripts. Because comparison of gene expression levels is frequently the aim of these experiments, there is a critical need to validate internal control genes. When studying gene expression in bovine polymorphonuclear leukocytes, special attention should be paid to this validation, because polymorphonuclear leukocytes are subjected to numerous physiological influences, depending on the stage of lactation. In this study, 8 commonly used reference genes (ACT, GAPD, H2A, TBP, HPRT1, SDHA, YWHAZ, and 18S rRNA) were evaluated in bovine polymorphonuclear leukocytes. The transcription levels of 6 reference genes were determined using real-time PCR. By geometrically averaging the expression levels of these genes, SDHA, YWHAZ, and 18S rRNA were selected as being the most stable genes for accurate normalization of real-time results of bovine polymorphonuclear leukocytes

Gene expression analysis in specific cell populations of bovine blastocysts using laser capture microdissection

Laser capture microdissection (LCM) has become a powerful technique for the isolation of specific cell populations from heterogeneous tissues for gene expression analysis. For standard laser capture microdissection procedures, snap freezing and cryosectioning is the optimal fixation and processing procedure. This sufficiently preserves RNA integrity for downstream molecular analysis. However, in the case of small sized samples, such as early stage embryos, snap freezing and cryosectioning is highly complex and frequently leads to the loss of valuable samples. In the present study, a protocol was optimized to specifically isolate the inner cell mass cells from bovine blastocysts with LCM on chemically fixated paraffin embedded embryos. Bovine blastocysts were fixed for 24h in a methacarn fixative (8 parts methanol, 1 part acetic acid), upon which they were embedded in 2% agarose. After paraffin embedding of the agarose blocks, serial sections of 10 µm were cut and adhered to glass slides. The slides were deparaffinized in xylene, stained with 0.1% cresyl violet in 85% ethanol, and dehydrated in xylene. Subsequently the trophectoderm cells and the inner cell mass cells were separately isolated using LCM. The RNA of the samples was isolated, and subjected to different tests to evaluate the purity of the samples and the quality of the RNA. Expression of Cytokeratin 18 which is only expressed in trophectoderm revealed that the isolated cells were highly pure. An amplicon length assay and a 3’-5’ assay revealed that the RNA quality was sufficient for gene expression analysis with RT-qPCR. In conclusion, LCM on blastocysts is well suited for gene expression analysis of specific cell populations, providing a novel strategy to investigate molecular pathways for pluripotency and cell fate development. This procedure is also compatible with micro-RNA profiling strategies, and the application for downstream transcriptome sequencing is currently being evaluated

Differences in brain processing of proprioception related to postural control in patients with recurrent non-specific low back pain and healthy controls

Patients with non-specific low back pain (NSLBP) show an impaired postural control during standing and a slower performance of sit-to-stand-to-sit (STSTS) movements. Research suggests that these impairments could be due to an altered use of ankle compared to back proprioception. However, the neural correlates of these postural control impairments in NSLBP remain unclear. Therefore, we investigated brain activity during ankle and back proprioceptive processing by applying local muscle vibration during functional magnetic resonance imaging in 20 patients with NSLBP and 20 controls. Correlations between brain activity during proprioceptive processing and (Airaksinen et al., 2006) proprioceptive use during postural control, evaluated by using muscle vibration tasks during standing, and (Altmann et al., 2007) STSTS performance were examined across and between groups. Moreover, fear of movement was assessed. Results revealed that the NSLBP group performed worse on the STSTS task, and reported more fear compared to healthy controls. Unexpectedly, no group differences in proprioceptive use during postural control were found. However, the relationship between brain activity during proprioceptive processing and behavioral indices of proprioceptive use differed significantly between NSLBP and healthy control groups. Activity in the right amygdala during ankle proprioceptive processing correlated with an impaired proprioceptive use in the patients with NSLBP, but not in healthy controls. Moreover, while activity in the left superior parietal lobule, a sensory processing region, during back proprioceptive processing correlated with a better use of proprioception in the NSLBP group, it was associated with a less optimal use of proprioception in the control group. These findings suggest that functional brain changes during proprioceptive processing in patients with NSLBP may contribute to their postural control impairments

Characterization and profiling of immunomodulatory genes of equine mesenchymal stromal cells from non-invasive sources

Introduction: Mesenchymal stromal cells (MSCs) have been extensively studied for their promising capabilities in regenerative medicine. Although bone marrow is the best-known source for isolating equine MSCs, non-invasive alternative sources such as umbilical cord blood (UCB), umbilical cord matrix (UCM), and peripheral blood (PB) have also been reported. Methods: Equine MSCs from three non-invasive alternative sources were isolated from six individual mares (PB) and their foals (UCB and UCM) at parturition. To minimize inter-horse variability, the samples from the three sources were matched within the same mare and for UCB and UCM even within the same foal from that specific mare. The following parameters were analyzed: (i) success rate of isolation, (ii) proliferation capacity, (iii) tri-lineage differentiation ability, (iv) immunophenotypical protein, and (v) immunomodulatory mRNA profiles. Linear regression models were fit to determine the association between the source of MSCs (UCB, UCM, PB) and (i) the moment of first observation, (ii) the moment of first passage, (iii) cell proliferation data, (iv) the expression of markers related to cell immunogenicity, and (v) the mRNA profile of immunomodulatory factors, except for hepatocyte growth factor (HGF) as no normal distribution could be obtained for the latter variable. To evaluate the association between the source of MSCs and the mRNA expression of HGF, the non-parametric Kruskal-Wallis test was performed instead. Results: While equine MSCs could be isolated from all the UCB and PB samples, isolation from UCM was successful in only two samples because of contamination issues. Proliferation data showed that equine MSCs from all three sources could be easily expanded, although UCB-derived MSCs appeared significantly faster in culture than PB- or UCM-derived MSCs. Equine MSCs from both UCB and PB could be differentiated toward the osteo-, chondro-, and adipogenic lineage, in contrast to UCM-derived MSCs in which only chondro-and adipogenic differentiation could be confirmed. Regardless of the source, equine MSCs expressed the immunomodulatory genes CD40, CD80, HGF, and transforming growth factor-beta (TGF beta). In contrast, no mRNA expression was found for CD86, indoleamine 2,3-dioxygenase (IDO), and tumor necrosis factor-alpha (TNF alpha). Conclusions: Whereas UCM seems less feasible because of the high contamination risks and low isolation success rates, UCB seems a promising alternative MSC source, especially when considering allogeneic MSC use

The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells

Quantification of Fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos

<p>Abstract</p> <p>Background</p> <p>Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.</p> <p>Results</p> <p>RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells.</p> <p>In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 α integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 β integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV).</p> <p>Conclusion</p> <p>The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.</p

Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A

BACKGROUND: An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor γ coactivator 1α (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. RESULTS: The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B), the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05). CONCLUSION: This study provides a new set of reference genes (ACTB, TBP and TOP2B) suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass

Identification and expression analysis of genes associated with bovine blastocyst formation

<p>Abstract</p> <p>Background</p> <p>Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation.</p> <p>First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of <it>in vitro </it>embryo production procedures.</p> <p>Results</p> <p>A subtractive cDNA library was constructed enriched for transcripts preferentially expressed at the blastocyst stage compared to the 2-cell and 8-cell stage. Sequence information was obtained for 65 randomly selected clones. The RNA expression levels of 12 candidate genes were determined throughout 3 stages of preimplantation embryo development (2-cell, 8-cell and blastocyst) and compared with the RNA expression levels of <it>in vivo </it>"golden standard" embryos using real-time PCR. The RNA expression profiles of 9 (75%) transcripts (<it>KRT18</it>, <it>FN1</it>, <it>MYL6</it>, <it>ATP1B3</it>, <it>FTH1</it>, <it>HINT1</it>, <it>SLC25A5</it>, <it>ATP6V0B</it>, <it>RPL10</it>) were in agreement with the subtractive cDNA cloning approach, whereas for the remaining 3 (25%) (<it>ACTN1</it>, <it>COPE</it>, <it>EEF1A1</it>) the RNA expression level was equal or even higher at the earlier developmental stages compared to the blastocyst stage. Moreover, significant differences in RNA expression levels were observed between <it>in vitro </it>and <it>in vivo </it>produced embryos. By immunofluorescent labelling, the protein expression of KRT18, FN1 and MYL6 was determined throughout bovine preimplantation embryo development and showed the same pattern as the RNA expression analyses.</p> <p>Conclusion</p> <p>By subtractive cDNA cloning, candidate genes involved in blastocyst formation were identified. For several candidate genes, important differences in gene expression were observed between <it>in vivo </it>and <it>in vitro </it>produced embryos, reflecting the influence of the <it>in vitro </it>culture system on the embryonic gene expression. Both RNA and protein expression analysis demonstrated that <it>KRT18</it>, <it>FN1 </it>and <it>MYL6 </it>are differentially expressed during preimplantation embryo development and those genes can be considered as markers for bovine blastocyst formation.</p

Regulatory microRNA network identification in bovine blastocyst development

Mammalian blastocyst formation is characterized by two lineage segregations resulting in the formation of the trophectoderm, the hypoblast, and the epiblast cell lineages. Cell fate determination during these early lineage segregations is associated with changes in the expression of specific transcription factors. In addition to the transcription factor-based control, it has become clear that also microRNAs (miRNAs) play an important role in the post-transcriptional regulation of pluripotency and differentiation. To elucidate the role of miRNAs in early lineage segregation, we compared the miRNA expression in early bovine blastocysts with the more advanced stage of hatched blastocysts. Reverse transcription-quantitative PCR-based miRNA expression profiling revealed eight upregulated miRNAs (miR-127, miR-130a, miR-155, miR-196a, miR-203, miR-28, miR-29c, and miR-376a) and four downregulated miRNAs (miR-135a, miR-218, miR-335, and miR-449b) in hatched blastocysts. Through an integrative analysis of matching miRNA and mRNA expression data, candidate miRNA-mRNA interaction pairs were prioritized for validation. Using an in vitro luciferase reporter assay, we confirmed a direct interaction between miR-218 and CDH2, miR-218 and NANOG, and miR-449b and NOTCH1. By interfering with the FGF signaling pathway, we found functional evidence that miR-218, mainly expressed in the inner cell mass, regulates the NANOG expression in the bovine blastocyst in response to FGF signaling. The results of this study expand our knowledge about the miRNA signature of the bovine blastocyst and of the interactions between miRNAs and cell fate regulating transcription factors