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 μg/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 high-quality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy.
Similar content being viewed by others
References
Bionaz M, Loor JJ (2007) Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during the lactation cycle. Physiol Genom 29:312–319
Farr VC, Stelwagen K, Cate LR, Molenaar AJ, McFadden TB, Davis SR (1996) An improved method for the routine biopsy of bovine mammary tissue. J Dairy Sci 79:543–549
Jenkins TC, McGuire MA (2006) Major advances in nutrition: impact on milk composition. J Dairy Sci 89:1302–1310
Soyeurt H, Dardenne P, Gillon A, Croquet C, Vanderick S, Mayeres P, Bertozzi C, Gengler N (2006) Variation in fatty acid contents of milk and milk fat within and across breeds. J Dairy Sci 89:4858–4865
Bionaz M, Loor JJ (2008) Gene networks driving bovine milk fat synthesis during the lactation cycle. BMC Genomics 9:366
Murrieta CM, Hess BW, Scholljegerdes EJ, Engle TE, Hossner KL, Mossand GE, Rule DC (2006) Evaluation of milk somatic cells as a source of mRNA for study of lipogenesis in the mammary gland of lactating beef cows supplemented with dietary high-linoleate safflower seeds. J Anim Sci 84:2399–2405
Boutinaud M, Rulquin H, Keisler DH, Djiane J, Jammes H (2002) Use of somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. J Anim Sci 80:1258–1269
Feng S, Salter AM, Parr T, Garnsworthy PC (2007) Extraction and quantitative analysis of stearoyl-coenzyme A desaturase mRNA from dairy cow milk somatic cells. J Dairy Sci 90:4128–4136
Dulin AM, Paape MJ, Berkow S, Hamosh M, Hamosh P (1983) Comparison of total somatic cells and differential cellular composition in milk from cows, sheep, goats, and humans. Fed Proc 42:1331
Boutinaud M, Jammes H (2002) Potential uses of milk epithelial cells: a review. Reprod Nutr Develop 42:133–147
Paape MJ, Wiggans GR, Bannermana DD, Thomasc DL, Sanders AH, Contreras A, Moroni P, Miller RH (2007) Monitoring goat and sheep milk somatic cell counts. Small Rum Res 68:114–125
Harvatine KJ, Bauman DE (2006) SREBP1 and thyroid hormone responsive spot 14 (S14) are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with CLA. J Nutr 136:2468–2474
Acknowledgments
This study was supported by funding from the Regione Autonoma della Sardegna (L.R. 7/2007).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mura, M.C., Daga, C., Bodano, S. et al. Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep. Mol Biol Rep 40, 2169–2173 (2013). https://doi.org/10.1007/s11033-012-2276-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-012-2276-6