اعتبارسنجی ژن‌های خانه‌دار جهت نرمال کردن Real Time PCR در سلول‌های سوماتیکی تلیسه‌ای شیری هلشتاین

Subject Areas : Camel

م. 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

Received: 2016-09-04 Accepted : 2017-01-31 Published : 2017-06-01

Keywords: BestKeeper, تلیسه‌های شیری, سلولهای سوماتیکی شیر, ژن مرجع,

Abstract :

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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