Validation of Reference Genes for Real Time PCR Normalization in Milk Somatic Cells of Holstein Dairy Cattle
محورهای موضوعی : Camelم. محقق-دولت‌آبادی 1 , ح. حسینی-دولت‌آبادی 2 , ا. حیدری ارجلو 3 , ر. محمودی 4
1 - Department of Animal Science, Faculty of Agriculture, Yasouj University, Yasouj, Iran
2 - Department of Animal Science, Faculty of Agriculture, Yasouj University, Yasouj, Iran
3 - Cellular and Molecular Research Center, Yasouj University of Medical Science, Yasouj, Iran
4 - Cellular and Molecular Research Center, Yasouj University of Medical Science, Yasouj, Iran
کلید واژه: dairy heifers, milk somatic cell, reference gene, best keeper,
چکیده مقاله :
Real time-qPCR is the most reliable method for evaluation of mRNA expression levels. However, to obtain accurate results, selection of suitable reference genes is necessary for normalizing the real-time qPCR data. The aim of this research was to validate the expression stability of three potential reference genes (ACTB, GAPDH and UXT) in milk somatic cells of Holstein dairy cattle under different lactation stages. For this purpose two types of milk samples from eighteen healthy cows at three lactation stages (early, middle and late of lactation cycle) and four mastitic cows were included in this experiment. Total RNA was extracted from the milk somatic cells and then cDNA was synthesized. Real-time polymerase chain reaction (PCR) performed for actb, gapdh and UXT genes as candidate reference genes. Then, the real-time PCR results were analyzed with BestKeeper program. The evaluation of selected genes by real-time PCR revealed that all genes were expressed in the healthy and mastitic dairy cows. In addition, the UXT and GADPH genes displayed the lowest and highest values of expression level, respectively. The ACTB gene was considered as the most suitable internal controls as it was stably expressed in milk somatic cells regardless of dairy cows conditions. Taken together, our results could help to select suitable reference gene for the normalization of expression levels in milk somatic cells of dairy cattle.
تکنیک Real time PCR مطمئن‌ترین روش ارزیابی سطوح بیان mRNA می‌باشد. اگرچه، جهت به دست آوردن نتایج دقیق، انتخاب ژن‌های مرجع مناسب جهت نرمال کردن داده‌هایReal time PCR ضروری است. هدف از این تحقیق بررسی پایداری بیان 3 ژن پتانسیل مرجع (ACTB، GAPDH و UXT) در سلول‌های سوماتیکی شیر تلیسه‌های شیری هلشتاین در شرایط متفاوت بود. برای این منظور، دو نوع نمونه شیر از 18 تلیسه شیری سالم در مراحل مختلف شیردهی و 4 تلیسه مبتلا به ورم پستان در این آزمایش در نظر گرفته شد. RNA کل از سلول‌های سوماتیکی استخراج شد و سپس cDNA سنتز شد. واکنش Real time PCR برای ژن‌های ACTB، GAPDHو UXT به عنوان ژن‌های خانه‌دار کاندیدا انجام شد و نتایج توسط برنامه BestKeepe مورد تجزیه و تحلیل قرار گرفت. ارزیابی ژن‌های توسط Real time PCR نشان داد که همه ژن ها در تلیسه های شیری سالم و مبتلا به ورم پستان بیان شده بودند. علاوه بر این، ژن‌های UXT و GAPDH به ترتیب دارای کمترین و بیشترین مقدار بیان بودند. ژن ACTB به عنوان مناسب‌ترین کنترل داخلی در نظر گرفته شد و در سلول‌های سوماتیکی صرف نظر از شرایط تلیسه‌های شیری دارای ثبات بیان بود. بر این اساس، نتایج مطالعه ما می‌تواند در انتخاب ژن مرجع مناسب برای نرمال کردن سطوح بیان در سلول‌های سوماتیکی شیر تلیسه‌های شیری کمک کند.
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