ارزیابی پایدارترین ژن‌های مرجع در بافت بیضه‌ی مردان آزواسپرم

نوع مقاله : مقاله های پژوهشی

نویسندگان

1 دانشجوی دکتری، گروه زیست‌شناسی، دانشکده‌ی علوم، دانشگاه اصفهان، اصفهان، ایران

2 دانشیار، گروه زیست‌شناسی، دانشکده‌ی علوم، دانشگاه اصفهان، اصفهان، ایران

3 دانشیار، گروه زیست‌شناسی، دانشکده‌ی علوم، دانشگاه اصفهان و پژوهشگاه رویان، پژوهشکده‌ی زیست فن‌آوری جهاد دانشگاهی، مرکز تحقیقات علوم سلولی و گروه زیست فن‌آوری مولکولی، دانشکده‌ی، دانشگاه، اصفهان، ایران

4 استاد، پژوهشگاه رویان، پژوهشکده‌ی زیست فن‌آوری جهاد دانشگاهی، مرکز تحقیقات علوم سلولی و مرکز باروری و ناباروری اصفهان (IFIC)، اصفهان، ایران

5 دانشجو، گروه ارولوژی، دانشکده‌ی پزشکی، دانشگاه علوم پزشکی اصفهان، اصفهان، ایران

چکیده

مقدمه: واکنش PCR کمی در زمان واقعی (Real-time quantitative reverse transcriptase-polymerase chain reaction یا qRT-PCR)، یک روش سریع، حساس و قابل اعتماد برای مقایسه‌ی بیان ژن‌ها می‌باشد که مستعد خطاهای تکنیکی فراوانی است. همچنین، استفاده از ژن‌های مرجع (خانه‌گردان) تاریخی، همیشه و در همه‌ی بافت‌ها مناسب نمی‌باشد. در این مطالعه، به بررسی و انتخاب ژن(های) مرجع مناسب در بافت بیضه پرداخته شد تا بتوان از این ژن(های) مناسب برای انجام واکنش qRT-PCR استفاده کرد.روش‌ها: نمونه‌ی بافت بیضه از 15 مرد آزواسپرم غیر انسدادی (NOA یا Non-obstructive azoospermia) به عنوان گروه مورد و 15 مرد آزواسپرم انسدادی (OA یا Obstructive azoospermia) به عنوان گروه شاهد گرفته شد. با استفاده از نرم‌افزار Beacon designer 8.1 پرایمرهای مناسب برای چهار ژن مرجع Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)، Ribosomal protein L37 (RPL37)، Ring finger protein 1 (RING1) و Eukaryotic translation elongation factor 2 (eEF2) طراحی شد. بهینه‌سازی‌های قبل از انجام qRT-PCR، شامل کنترل مقدار RNA برای سنتز Complementary DNA (cDNA) و تعیین غلظت مناسب پرایمرها انجام شد. منحنی ذوب رسم گردید و مقادیر Quantitation cycle (Cq) استخراج شد. آنالیز میانگین Cq در دو گروه شاهد و مورد با استفاده از نرم‌افزار BestKeeper نسخه‌ی 1 انجام شد و ژن‌های مرجع مناسب انتخاب گردید.یافته‌ها: مقایسه‌ی میانگین Cq بین دو گروه NOA و OA حاکی از آن بود که دو ژن RPL37 و GAPDH به ترتیب با انحراف معیار 39/1 و 67/1 کمترین تغییرات را در بین چهار ژن مرجع کاندیدا نشان داد. بنا بر این، دو ژن RPL37 و GAPDH، به ترتیب با مقادیر r برابر با 959/0 و 927/0، به عنوان مناسب‌ترین ژن‌های مرجع در بافت بیضه انتخاب شد.نتیجه‌گیری: دو ژن RPL37 و GAPDH به ترتیب، مناسب‌ترین ژن‌های مرجع در بافت بیضه هستند و برای مطالعه‌ی بیان ژن‌ها با استفاده از روش qRT-PCR مناسب می‌باشند.

کلیدواژه‌ها

عنوان مقاله [English]

Evaluation of the Most Stable Reference Genes in Testicular Tissue of the Men with Azoospermia

نویسندگان [English]

  • Seyed Morteza Javadirad 1
  • Zohreh Hojati 2
  • Kamran Ghaedi 3
  • Mohammad Hossein Nasr-Esfahani 4
  • Behzad Abbasy 5
1 PhD Student, Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran
2 Associate Professor, Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran
3 Associate Professor, Department of Biology, School of Sciences, University of Isfahan AND Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, Academic Center for Education, Culture and Research (ACER), Isfahan, Iran
4 Professor, Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, Academic Center for Education, Culture and Research (ACER) AND Isfahan Fertility and Infertility Center (IFIC), Isfahan, Iran
5 Student, Department of Orology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
چکیده [English]

Background: Real-time quantitative reverse transcriptase- polymerase chain reaction (qRT-PCR), is a fast, sensitive and reliable method of gene expression comparison that is prone to a lot of technical errors. On the other hand, historical reference (housekeeping) genes are not suitable for all tissues. Herein, we have tried to identify and evaluate the best reference gene for testis tissues for further qRT-PCR experiments.Methods: Testis tissues of 15 men with non-obstructive (NOA) and 15 men with obstructive (OA) azoospermia (as control individuals) were collected. Primer designing and verification of four candidate reference genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L37 (RPL37), ring finger protein 1 (RING1) and eukaryotic translation elongation factor 2 (eEF2) were performed using Beacon designer 8.1 software. PCR pre-optimization for reverse transcriptase input RNA and best primer concentration were included. Melt curve analysis was drawn and values of quantitation cycle (Cq) were extracted. Mean Cq analysis was calculated using BestKeeper v1 software and suitable reference genes were selected afterward.Findings: Comparing the mean Cq values between the NOA and OA groups declared that RPL37 and GAPDH showed the lowest standard deviations of 1.39 and 1.67 among the other candidates. GAPDH and RPL37 were selected as the best reference genes in testis tissues with their r values of 0.959 and 0.927, respectively.Conclusion: The results of this study show that the best reference genes for normalization of qRT-PCR data of testis tissues are GAPDH and RPL37.

کلیدواژه‌ها [English]

  • Reference gene
  • Testis
  • Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
  • BestKeeper software

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مجله دانشکده پزشکی اصفهان
دوره 33، شماره 367 - شماره پیاپی 367
بهمن و اسفند 1394
صفحه 2407-2416

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سابقه مقاله

  • تاریخ دریافت: 20 مهر 1394
  • تاریخ بازنگری: 01 اردیبهشت 1403
  • تاریخ پذیرش: 17 فروردین 1401

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