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Is total RNA extraction possible from small volume goat milk (7.5 mL) for RT-qPCR studies?

Yıl 2018, , 57 - 61, 01.03.2018

Aysel Şakar Eraslan Hüseyin Özkan Akın Yakan

https://doi.org/10.1501/Vetfak_0000002827

Öz

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

Anahtar Kelimeler

Goat milk volume RNA extraction RT-qPCR

Kaynakça

  • 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.

RT-qPCR çalışmaları için küçük hacimli keçi sütlerinden (7.5 mL) total RNA ekstraksiyonu mümkünmüdür?

Yıl 2018, , 57 - 61, 01.03.2018

Aysel Şakar Eraslan Hüseyin Özkan Akın Yakan

https://doi.org/10.1501/Vetfak_0000002827

Öz

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

Anahtar Kelimeler

Keçi RNA ekstraksiyonu RT-qPCR süt hacmi

Kaynakça

  • 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.
Toplam 20 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Veteriner Cerrahi
Diğer ID JA64AN54RH
Bölüm Araştırma Makalesi
Yazarlar

Aysel Şakar Eraslan

Hüseyin Özkan

Akın Yakan

Yayımlanma Tarihi 1 Mart 2018
Yayımlandığı Sayı Yıl 2018

Kaynak Göster

APA Eraslan, A. Ş., Özkan, H., & Yakan, A. (2018). Is total RNA extraction possible from small volume goat milk (7.5 mL) for RT-qPCR studies?. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 65(1), 57-61. https://doi.org/10.1501/Vetfak_0000002827
AMA Eraslan AŞ, Özkan H, Yakan A. Is total RNA extraction possible from small volume goat milk (7.5 mL) for RT-qPCR studies?. Ankara Univ Vet Fak Derg. Mart 2018;65(1):57-61. doi:10.1501/Vetfak_0000002827
Chicago Eraslan, Aysel Şakar, Hüseyin Özkan, ve Akın Yakan. “Is Total RNA Extraction Possible from Small Volume Goat Milk (7.5 ML) for RT-QPCR Studies?”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65, sy. 1 (Mart 2018): 57-61. https://doi.org/10.1501/Vetfak_0000002827.
EndNote Eraslan AŞ, Özkan H, Yakan A (01 Mart 2018) Is total RNA extraction possible from small volume goat milk (7.5 mL) for RT-qPCR studies?. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65 1 57–61.
IEEE A. Ş. Eraslan, H. Özkan, ve A. Yakan, “Is total RNA extraction possible from small volume goat milk (7.5 mL) for RT-qPCR studies?”, Ankara Univ Vet Fak Derg, c. 65, sy. 1, ss. 57–61, 2018, doi: 10.1501/Vetfak_0000002827.
ISNAD Eraslan, Aysel Şakar vd. “Is Total RNA Extraction Possible from Small Volume Goat Milk (7.5 ML) for RT-QPCR Studies?”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65/1 (Mart 2018), 57-61. https://doi.org/10.1501/Vetfak_0000002827.
JAMA Eraslan AŞ, Özkan H, Yakan A. Is total RNA extraction possible from small volume goat milk (7.5 mL) for RT-qPCR studies?. Ankara Univ Vet Fak Derg. 2018;65:57–61.
MLA Eraslan, Aysel Şakar vd. “Is Total RNA Extraction Possible from Small Volume Goat Milk (7.5 ML) for RT-QPCR Studies?”. Ankara Üniversitesi Veteriner Fakültesi Dergisi, c. 65, sy. 1, 2018, ss. 57-61, doi:10.1501/Vetfak_0000002827.
Vancouver Eraslan AŞ, Özkan H, Yakan A. Is total RNA extraction possible from small volume goat milk (7.5 mL) for RT-qPCR studies?. Ankara Univ Vet Fak Derg. 2018;65(1):57-61.