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

Abstract

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

Keywords

• Goat
• milk volume
• RNA extraction
• RT-qPCR

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

Abstract

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

Keywords

• Keçi
• RNA ekstraksiyonu
• RT-qPCR
• süt hacmi

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