ankara univ vet fak derg Ankara Üniversitesi Veteriner Fakültesi Dergisi 1300-0861 1308-2817 Ankara University 10.1501/Vetfak_0000002827 Veterinary Surgery Veteriner Cerrahi Is total RNA extraction possible from small volume goat milk (7.5 mL) for RT-qPCR studies? RT-qPCR çalışmaları için küçük hacimli keçi sütlerinden (7.5 mL) total RNA ekstraksiyonu mümkünmüdür? Eraslan Aysel Şakar Özkan Hüseyin Yakan Akın 03 01 2018 65 1 57 61 03 01 2018 Copyright © 1954, Ankara Üniversitesi Veteriner Fakültesi Dergisi 1954 Ankara Üniversitesi Veteriner Fakültesi Dergisi

This study aimed to study the application availability of RT-qPCR by total RNA extraction from small volumes of milk samples. RNA was extracted from 7,5 mL milk. Amount and quality of RNA and Ct results obtained via RT-qPCR were compared with the values obtained from 100 mL milk. A260/A280 ratio of absorbance was found to be higher than 1,70 in all samples. Mean RNA purity were identified as 1,87±0,07 and 1,77±0,01 in 7,5 mL and 100 mL milk samples respectively. While mean total RNA concentration was 149,37±43,40 µg/mL in 7,5 mL milk sample, it was measured to be 309,03±77,82 µg/mL in 100 mL milk sample. Mean somatic cell count (SCC) of the 8 goats used in the study was calculated as 486,37±185,68 x 103 cells/mL. Ribosomal Protein Lateral Stalk Subunit P0 (RPLP0) gene which is accepted as the reference gene in goat somatic cells was used to test application availability of obtained RNA in RT-qPCR. Mean Ct results were found to be 20,33±0,39 and 19,51±0,41 in 7,5 mL and 100 mL groups respectively, creating no difference between the two groups (P = 0,170). Quality and amount of obtained RNA and Ct results showed that it was possible to extract RNA by using 7,5 mL milk

Bu çalışmada küçük hacimlerdeki süt örneklerinden total RNA izolasyonu yapılarak, RT-qPCR çalışmalarının yapılabilirliğinin araştırılması amaçlanmıştır. Bunun için 7,5 mL sütten RNA izole edilmiş; RNA miktarı, kalitesi ve RT-qPCR’dan elde edilen Ct sonuçları 100 mL sütten elde edilen değerlerle karşılaştırılmıştır. Kullanılan bütün örneklerde A260/A280 absorbans oranı 1,70’ten büyük, RNA saflığının ortalaması 7,5 mL örnek grubunda 1,87±0,07, 100 mL örnek grubunda 1,77±0,01 olarak tespit edilmiştir. Total RNA konsantrasyonunun ortalaması 7,5 mL örnek grubunda 149,37±43,40 µg/mL iken, 100 mL örnek grubunda 309,03±77,82 µg/mL olarak ölçülmüştür. Kullanılan 8 keçinin somatik hücre sayısının (SCC) ortalaması ise 486,37±185,68 x 103olarak hesaplanmıştır. Elde edilen RNA’ların RT-qPCR’da kullanılabilirliğini test etmek için keçi süt somatik hücrelerinde referans gen olarak kabul edilen Ribosomal Protein Lateral Stalk Subunit P0 (RPLP0) geni kullanılmıştır. 7,5 mL grubunda Ct sonuçlarının ortalaması 20,33±0,39, 100 mL grubunda 19,51±0,41 olarak bulunmuş olup, iki grup arasında bir fark oluşmamıştır (P=0,170). Elde edilen RNA’nın miktarı, kalitesi ve Ct sonuçları 7,5 mL süt kullanılarak da RNA izolasyonu yapılabileceğini göstermiştir

 Goat milk volume RNA extraction RT-qPCR Keçi RNA ekstraksiyonu RT-qPCR süt hacmi Ausubel FM, Brent R, Kingston RE, et al. (2003): Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Boston. Boutinaud M, Rulquin H, Keisler DH, Djiane J, et al. (2002): Use of somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. J Anim Sci, 80, 1258-1269. Ceballos LS, Morales ER, de la Torre Adarve G, et al. (2009): Composition of goat and cow milk produced under similar conditions and analyzed by identical methodology. J Food Compos Anal, 22, 322-329. Cremonesi P, Capoferri R, Pisoni G, et al. (2012): Response of the goat mammary gland to infection with Staphylococcus aureus revealed by gene expression profiling in milk somatic and white blood cells. BMC genomics, 13, 1. Fao (2015): Available at http://faostat3.fao.org/browse/Q/ QL/E. (Accessed July 18, 2016). Farrell RE (2005): RNA Methodologies: A Laboratory Guide for Isolation and Characterization. 3rd ed., Elsevier Academic Press Inc., USA. Feng S, Salter AM, Parr T, et al. (2007): Extraction and quantitative analysis of stearoyl-coenzyme A desaturase mRNA from dairy cow milk somatic cells. J Dairy Sci, 90, 4128-4136. Finot L, Marnet PG, Dessauge F (2011): Reference gene selection for quantitative real-time PCR normalization: application in the caprine mammary gland. Small Ruminant Res, 95, 20-26. Fleige S, Pfaffl MW (2006): RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med, 27, 126-139. Jarczak J, Kaba J, Bagnicka E (2014): The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis: application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus. Gene, 549, 280-285. Lemay DG, Hovey RC, Hartono SR, et al. (2013): Sequencing the transcriptome of milk production: milk trumps mammary tissue. BMC genomics, 14, 1. Mestawet TA, Girma A, Adnoy T, et al. (2013): Newly identified mutations at the CSN1S1 gene in Ethiopian goats affect casein content and coagulation properties of their milk. J Dairy Sci, 96, 4857-4869. Mura MC, Daga C, Bodano S, et al. (2013): Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep. Mol Biol Rep, 40, 2169-2173. Murrieta CM, Hess BW, Scholljegerdes EJ, et al. (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. Paape MJ, Capuco AV (1997): Cellular defense mechanisms in the udder and lactation of goats. J Anim Sci, 75, 556-565. Paape MJ, Poutrel B, Contreras A, et al. (2001): Milk somatic cells and lactation in small ruminants. J Dairy Sci, 84, E237-E244. Pfaffl MW, Wittmann SL, Meyer HHD, et al. (2003): Gene expression of immunologically important factors in blood cells, milk cells, and mammary tissue of cows. J Dairy Sci, 86, 538-545. Raynal-Ljutovac K, Lagriffoul G, Paccard P, et al. (2008): Composition of goat and sheep milk products: An update. Small Ruminant Res, 79, 57-72. Sarıkaya H (2006): Somatic cell populations in milk: Importance in mammary gland physiology and behaviour during technological processing. PhD Thesis. Technische Universität München, München. Varshney N, Mohanty AK, Kumar S, et al. (2012): Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus). J Dairy Sci, 95, 2935-2945.