Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep
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Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep Maria Consuelo Mura • Cinzia Daga • Sara Bodano • Marta Paludo • Sebastiano Luridiana • Michele Pazzola • Maria Luisa Dettori • Giuseppe Massimo Vacca Vincenzo Carcangiu
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Received: 21 August 2012 / Accepted: 19 November 2012 / Published online: 24 November 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract The aim of the study was to develop a reliable method for the RNA extraction from milk of Sarda sheep breed and to highlight if the extracted RNA can be used for expression study on mammary genes involved in milk fat synthesis using RT-qPCR. The main result is that a sample of 150 ml of milk provides an optimal amount of RNA (73.5 lg/ml). The highest RNA concentration has been found in the samples analysed within 4 h after collection. The RNA extracted was positively correlated to the number of somatic cells (P \ 0.001). The efficiency of the extraction method was confirmed by the results obtained from qPCR which showed a Ct value, for SREBPF1 gene of 26.8 ± 0.15. This research demonstrated that the highquality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy. Keywords Sheep Milk somatic cell RNA extraction Gene expression
Introduction The study of the mechanisms involved in the regulation of milk secretion has always attracted considerable interest because it could provide useful information for improving the quality of mammary secretion [1]. The identification of the mechanism involved in the milk secretion and the study of genes regulating these processes remain key-steps in the
M. C. Mura C. Daga S. Bodano M. Paludo S. Luridiana M. Pazzola M. L. Dettori G. M. Vacca V. Carcangiu (&) Dipartimento di Medicina Veterinaria, Universita` degli Studi di Sassari, Via Vienna 2, 07100 Sassari, Italy e-mail: [email protected]
selection plans aimed to improve productions in dairy mammals [2]. The rise in quality and quantity of the food supply is undoubtedly necessary to increase milk yield but it can only marginally affect its quality [3]. Contrarily, the identification of the genes involved in the regulation of milk secretion allows to manage quality improvement and to fix favourable genes [4]. To perform these studies is necessary to use modern molecular biology techniques which allows to highlights genes involved in milk secretion [5]. The first step to obtain RNA usually is a mammary gland biopsy, but this technique is particularly invasive and it can cause damage and infection [6]. Current methods have been developed for the RNA isolation from milk samples of goats [7] and cows [6, 8], but no work has been done yet on ewes milk. Thus the aim of the present study was to develop a reliable method for the total RNA extraction from Sarda breed sheep whole milk. The research has been conducted to identify the best condition for transport and storage of the milk samples, to establish the minimum
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