Dataset Information

Differential expression of CXCR1 and commonly used reference genes in bovine milk somatic cells following experimental intramammary challenge.

ABSTRACT: Chemokine (C-X-C motif) receptor 1 (CXCR1 or IL-8RA) plays an important role in the bovine mammary gland immunity. Previous research indicated polymorphism c.980A > G in the CXCR1 gene to influence milk neutrophils and mastitis resistance. In the present study, four c.980AG heifers and four c.980GG heifers were experimentally infected with Staphylococcus chromogenes. RNA was isolated from milk somatic cells one hour before and 12 hours after the experimental intramammary challenge. Expression of CXCR1 and eight candidate reference genes (ACTB, B2M, H2A, HPRT1, RPS15A, SDHA, UBC and YWHAZ) was measured by reverse transcription quantitative real-time PCR (RT-qPCR). Differences in relative CXCR1 expression between c.980AG heifers and c.980GG heifers were studied and the effect of the experimental intramammary challenge on relative expression of CXCR1 and the candidate reference genes was analyzed.Relative expression of CXCR1 was not associated with polymorphism c.980A > G but was significantly upregulated following the experimental intramammary challenge. Additionally, differential expression was detected for B2M, H2A, HPRT1, SDHA and YWHAZ.This study reinforces the importance of CXCR1 in mammary gland immunity and demonstrates the potential effect of experimental intramammary challenge on expression of candidate reference genes in milk somatic cells.

SUBMITTER: Verbeke J

PROVIDER: S-EPMC4421990 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Differential expression of CXCR1 and commonly used reference genes in bovine milk somatic cells following experimental intramammary challenge.

Verbeke Joren J Van Poucke Mario M Peelman Luc L De Vliegher Sarne S

BMC genetics 20150422

<h4>Background</h4>Chemokine (C-X-C motif) receptor 1 (CXCR1 or IL-8RA) plays an important role in the bovine mammary gland immunity. Previous research indicated polymorphism c.980A > G in the CXCR1 gene to influence milk neutrophils and mastitis resistance. In the present study, four c.980AG heifers and four c.980GG heifers were experimentally infected with Staphylococcus chromogenes. RNA was isolated from milk somatic cells one hour before and 12 hours after the experimental intramammary chall ...[more]

PMID: 25895496

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Project description:The aim of this study was to quantify some environmental (individual herds, herd productivity, milking system, and season) and animal factors [individual animals, breed, days in milk (DIM) and parity] on the variability of the log-10 transformation of somatic cell count (LSCC) and differential somatic cell count (DSCC) on individual bovine milk. A total of 159,360 test-day records related to milk production and composition were extracted from 12,849 Holstein-Friesian and 9,275 Simmental cows distributed across 223 herds. Herds were classified into high and low productivity, defined according to the average daily milk net energy output (DMEO) yielded by the cows. Data included daily milk yield (DYM; kg/d), milk fat, protein, lactose, SCC, and DSCC, and information on herds (i.e., productivity, milking system). The daily production of total and differential somatic cells in milk was calculated and then log-10 transformed, obtaining DLSCC and DLDSCC, respectively. Data were analyzed using a mixed model including the effects of individual herd, animal, repeated measurements intra animal as random, and herd productivity, milking system, season, breed, DIM, parity, DIM × parity, breed × season, DIM × milking system and parity × milking system as fixed factors. Herds with a high DMEO were characterized by a lower content of LSCC and DSCC, and higher DLSCC and DLDSCC, compared to the low DMEO herds. The association between milking system and somatic cell traits suggested that the use of the automatic milking systems would not allow for a rapid intervention on the cow, as evidenced by the higher content of all somatic cell traits compared to the other milking systems. Season was an important source of variation, as evidenced by high LSCC and DSCC content in milk during summer. Breed of cow had a large influence, with Holstein-Friesian having greater LSCC, DSCC, DLSCC, and DLDSCC compared to Simmental. With regard to DIM, the variability of LSCC was mostly related to that of DSCC, showing an increase from calving to the end of lactation, and suggesting the higher occurrence of chronic mastitis in cows toward the end of lactation. All the somatic cell traits increased across number of parities, possibly because older cows may have increased susceptibility to intramammary infections.

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.

Dataset Information

Differential expression of CXCR1 and commonly used reference genes in bovine milk somatic cells following experimental intramammary challenge.

Publications

Differential expression of CXCR1 and commonly used reference genes in bovine milk somatic cells following experimental intramammary challenge.

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