An optimized method of RNA isolation from goat milk somatic cells for transcriptomic analysis

Open access


The goat (Capra hircus) is a perfect animal model for analyzing the transcriptome of milk somatic cells (MSCs), as sufficient numbers of somatic cells in goat milk, i.e., exfoliated epithelial cells, can be obtained using noninvasive methods. RNA integrity and purity are the first and most important parameters qualifying samples for transcriptomic tests and next-generation sequencing, as RNA quality influences experimental results. The aim of this study was to optimize a method for obtaining high-quality RNA from goat MSCs, irrespective of effects like breed, lactation stage, health status (e.g., with or without small ruminant lentivirus [SRLV] infection), or number of somatic cells. Milk samples were obtained from goats of two Polish breeds in various lactation stages and in different parities, and from goats infected and not infected with SRLV. Altogether, 412 MSC samples were examined: 206 using method A with fenozol and 206 using method B with QIAzol. Though the overall purity (measured as absorbance ratios at 260 nm/280 nm and 260 nm/230 nm) of the RNA material was comparable, the average yield of RNA isolated using method A was 11.9 µg, while method B’s average yield was 29.9 µg. Moreover, method B resulted good quality RNA suitable for transcriptome analysis. Results were confirmed by RT-qPCR, using 18S rRNA and RPLP0 as the reference genes. The application of our modified treatment method was successful in obtaining high-integrity samples for transcriptomic or next-generation sequencing analysis. Using a 400 mL milk sample cooled in ice directly after milking, securing the cooling chain process from milking to MSC isolation, and applying method B to isolate RNA, we obtained good RNA quality irrespective of the goats’ breed, lactation stage, parity, milk yield, SRLV infection, and even milk yield and number of somatic cells in milk.

Badrul H.Y., McLachlan G., Vrettou C., Collie D. (2011). Optimising the quality and integrity of RNA samples from bronchial air way tissues. Asia Pac. J. Mol. Biol. Biotechnol., 19: 19–27.

Bagnicka E., Winnicka A., Jóźwik A., Rzewuska M., Strzałkowska N., Kościuczuk E., Prusak B., Kaba J., Horbańczuk J.O., Krzyżewski J. (2011). Relationship between somatic cell count and bacterial pathogens in goat milk. Small Rumin. Res., 100: 72-77.

Ben Chedly H., Boutinaud M., Bernier-Dodier P., Marnet P. G., Lacasse P. (2010). Disruption of cell junctions induces apoptosis and reduces synthetic activity in lactating goat mammary gland. J. Dairy Sci., 93: 2938–2951.

Ben Chedly H., Lacasse P., Marnet P., Komara M., Marion S., Boutinaud M. (2011). Use of milk epithelial cells to study regulation of cell activity and apoptosis during once-daily milking in goats. Animal 5(4): 572-579.

Boutinaud M., Galio L., Lollivier V., Finot L., Wiart S., Esquerré D., Devinoy E. (2013). Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling. Physiol. Genomics. 45(20): 973-985.

Boutinaud M., Jammes H. (2002a). Potential uses of milk epithelial cells: a review. Reprod. Nutr. Dev. 42(2): 133-147.

Boutinaud M., Rulquin H., Keisler D.H., Djiane J., Jammes H. (2002b) Use of somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. J. Anim. Sci. 80:1258–1269.

Brenaut P., Lefèvre L., Rau A., Laloë D., Pisoni G., Moroni P., Bevilacqua C., Martin P. (2014). Contribution of mammary epithelial cells to the immune response during early stages of a bacterial infection to Staphylococcus aureus. Vet. Res. 45:16.

Brown R., Epis M.R., Horsham J.L., Kabir T.D., Richardson K.L., Leedman P.J. (2018). Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal. BMC Biotechnol. 18(1):16.

Capuco A.V., Wood D.L., Baldwin R., McLeod K., Paape M.J. (2001). Mammary cell number, proliferation, and apoptosis during a bovine lactation: relation to milk production and effect of bST. J. Dairy Sci. 84(10): 2177-2187.

Cicinnati V.R., Shen Q., Sotiropoulos G.C., Radtke A., Gerken G., Beckebaum S. (2008). Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR. BMC Cancer 8: 350.

Cieslak J., Mackowski M., Czyzak-Runowska G., Wojtowski J., Puppel K., Kuczynska B., Pawlak P. (2015). Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells. PLoS ONE 10(10): e0139688.

Coombes B.K., Hardwidge P.R., Finlay B.B. (2004). Interpreting the Host-Pathogen Dialogue Through Microarrays. Adv. Appl. Microbiol. 54: 291-331.

Dalaly B.K., Eitenmiller R.R., Friend B.A., Shahani K.M. (1980). Human milk ribonuclease. Biochim. Biophys. Acta 65(2): 381–391.

Dastgheib S., Irajie C., Assaei R., Koohpeima F., Mokarram P. (2014). Optimization of RNA extraction from rat pancreatic tissue. Iran. J. Med. Sci. 39(3): 282-288.

Dudemaine P.L., Fomenky B., Dutoit A., Béjanin L., Ibeagha-Awemu E.M. (2017). 341 Comparison of five commercial kits for total RNA isolation including microRNA from three bovine milk fractions. J. Anim. Sci. 95(4): 168–169.

Finot L., Marnet P.G. and Dessauge F. (2011). Reference gene selection for quantitative real-time PCR normalization: application in the caprine mammary gland. Small Rumin. Res. 95(1): 20-26.

Fleige S., Pfafl M.W. (2006). RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects Med. 27(2-3): 126-139.

Fordyce, S.L., Kampmann, M.-L., van Doorn, N.L., Gilbert, M.T.P. (2013). Long-term RNA persistence in postmortem contexts. Investig. Genet. 4(1): 7.

Gallego Romero I., Pai A.A., Tung J., Gilad, Y. (2014). RNA-seq: impact of RNA degradation on transcript quantification. BMC Biol. 12: 42.

Gupta N., Mathur, M.P. (1989) Level and distribution of ribonuclease in milk from different species. Indian J. Dairy Sci. 42(3), 547-549.

Kolijn K., Geert J.L., van Leenders H. (2016) Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue. BMC Res. Not. 9: 7.

Kowalski M. (2009) Normy żywienia kóz mlecznych. W: IZ PIB-INRA. Normy żywienia przeżuwaczy: wartość pokarmowa francuskich i krajowych pasz dla przeżuwaczy[In Polish], (Standard of dairy goats’ feeding. In: NRIAP-INTA, Standard of ruminants’ feeding: nutrient value of French and domestic fodders for ruminants) Strzelecki (Ed.). Research Institute of Animal Production, Cracow, Poland, pp.109-119.

Kuang J., Yan X., Genders A.J., Granata C., Bishop D.J. (2018). An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research. PLoS ONE 13(5): e0196438.

Li R., Richoux N., Boutinaud M., Martin P., Gagnaire V. (2014). Role of somatic cells on dairy processes and products: a review. Dairy Sci Technol. 94(6): 517-538.

Li R., Dudemaine P.L., Zhao X., Lei C., Ibeagha-Awemu, E.M. (2016). Comparative Analysis of the miRNome of Bovine Milk Fat, Whey and Cells. PloS One 11(4): e0154129.

Livak K.J, Schmittgen T.D. (2001). Analysis of relative gene expression data using real time quantitative PCR and the 2∆∆C(T) Method. Methods 25(4): 402–408.

Majewska A., Domoradzki T., Grzelkowska-Kowalczyk K. (2019). Transcriptomic Profiling During Myogenesis. Methods Mol. Biol. 1889: 127-168.

Narrandes S., Xu W. (2018). Gene Expression Detection Assay for Cancer Clinical Use. J. Cancer 9(13): 2249-2265.

Norollahi S.A., Kokhaee P., Rashidy-Pour A., Hojati V., Norollahi S.E., Larijani L.V., Samadan A.A. (2018). Comparison of Methods of RNA Extraction From Breast and Gastric Cancer Tissues. Crescent J. Med. Biol. Sci. 5(1): 25–28.

Peirson S.N, Butler J.N. (2007). RNA extraction from mammalian tissues. Methods Mol. Biol. 362: 315-327

Pławińska-Czarnak J., Bagnicka E., Kaba J., Bogdan J., Zarzyńska J. (2014). Analysis of the CAEV infection impact on the milk yield and milk SCC of Polish dairy goats. J. Microbiol. Biotech. Food Sci. 3: 39-42.

Pławińska-Czarnak J., Zarzyńska J., Majewska A., Jank M., Kaba J., Bogdan J., Anusz K., Bagnicka E. (2019). Selected tissues of two Polish goat breeds do not differ on genomic level. Anim. Sci. Pap. Rep. 37 (1): 53-64.

Reiman M., Laan M., Rull K., Sõber S. (2017). Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples. FASEB J. 31(8): 3298-3308.

Reisberg S., Krebs K., Lepamets M., Kals M., Mägi R., Metsalu K., Lauschke V.M., Vilo J., Milani L. (2018) Translating genotype data of 44,000 biobank participants into clinical pharmacogenetic recommendations: challenges and solutions. Genet. Med.

Samadani, A.A., Nikbakhsh, N., Fattahi, S., Pourbagher, R., Aghajanpour Mir, S.M., Mousavi Kani, N., Abedian, Z., Akhavan-Niaki, H. (2015). RNA Extraction from Animal and Human’s Cancerous Tissues: Does Tissue Matter?. Int. J. Mol. Cel. Med. 4(1): 54-59.

Sanchez-Macias D., Castro N., Moreno-Indias I., Morales-delaNuez A., Briggs H., Capote J., Argüello A. (2010). The effects of storage temperature on goat milk somatic cell count using the DeLaval counter. Trop Anim Health Prod. 42(7): 1317-1320.

Schroeder A., Mueller O., Stocker S., Salowsky R., Leiber M., Gassmann M., Lightfoot S., Menzel W., Granzow M., Ragg T. (2006). The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 7: 3.

Sellin Jeffries M.K., Kiss A.J., Smith A.W., Oris, J.T. (2014). A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. BMC Biotechnol. 14: 94.

Suárez-Vega A., Gutiérrez-Gil B., Klopp C., Robert-Granie C., Tosser-Klopp G., Arranz J.J. (2015). Characterization and comparative analysis of the milk transcriptome in two dairy sheep breeds using RNA sequencing. Sci. Rep. 5: 18399.

Thompson K.L., Pine P.S., Rosenzweig B.A., Turpaz Y., Retief J. (2007). Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA. BMC Biotechnol. 7: 57.

Toral P.G., Hervás G., Suárez-Vega A., Arranz J.J., Frutos P. (2016). Isolation of RNA from milk somatic cells as an alternative to biopsies of mammary tissue for nutrigenomic studies in dairy ewes. J. Dairy Sci. 99(10): 8461-8471.

Wickramasinghe S., Rincon G., Islas-Trejo A., Medrano J.F. (2012). Transcriptional profiling of bovine milk using RNA sequencing. BMC Genomics. 13: 45.

Annals of Animal Science

The Journal of National Research Institute of Animal Production

Journal Information

IMPACT FACTOR 2017: 1.018
5-year IMPACT FACTOR: 0.959

CiteScore 2018: 1.4

SCImago Journal Rank (SJR) 2018: 0.509
Source Normalized Impact per Paper (SNIP) 2018: 0.869


All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 127 127 27
PDF Downloads 72 72 20