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

مراجع

  1. Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002; 30(6): 503-12.
  2. Vandesompele J, De PK, Pattyn F, Poppe B, Van Roy N, De PA, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3(7): RESEARCH0034.
  3. Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 2004; 37(1): 112-9.
  4. Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 2005; 6(4): 279-84.
  5. Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 2005; 344(1): 141-3.
  6. Arukwe A. Toxicological housekeeping genes: do they really keep the house? Environ Sci Technol 2006; 40(24): 7944-9.
  7. Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, Griffiths LR. Beta-actin--an unsuitable internal control for RT-PCR. Mol Cell Probes 2001; 15(5): 307-11.
  8. Tanic N, Perovic M, Mladenovic A, Ruzdijic S, Kanazir S. Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus-evaluation by real time RT-PCR. J Mol Neurosci 2007; 32(1): 38-46.
  9. Thellin O, Zorzi W, Lakaye B, De BB, Coumans B, Hennen G, et al. Housekeeping genes as internal standards: use and limits. J Biotechnol 1999; 75(2-3): 291-5.
  10. Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, et al. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 2002; 309(2): 293-300.
  11. Valenti MT, Bertoldo F, Dalle CL, Azzarello G, Zenari S, Zanatta M, et al. The effect of bisphosphonates on gene expression: GAPDH as a housekeeping or a new target gene? BMC Cancer 2006; 6: 49.
  12. Xu Z, Xu J, Ji A, Zhu Y, Zhang X, Hu Y, et al. Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum. Gene 2015; 574(2): 352-8.
  13. Kasioulis I, Syred HM, Tate P, Finch A, Shaw J, Seawright A, et al. Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis. Mol Biol Cell 2014; 25(8): 1216-33.
  14. Okada Y, Tateishi K, Zhang Y. Histone demethylase JHDM2A is involved in male infertility and obesity. J Androl 2010; 31(1): 75-8.
  15. Okada Y, Scott G, Ray MK, Mishina Y, Zhang Y. Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis. Nature 2007; 450(7166): 119-23.
  16. Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics 2005; 21(3): 389-95.
  17. Lin J, Redies C. Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression. Dev Genes Evol 2012; 222(6): 369-76.
  18. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009; 55(4): 611-22.
  19. Pansa A, Sirchia SM, Melis S, Giacchetta D, Castiglioni M, Colapietro P, et al. ESX1 mRNA expression in seminal fluid is an indicator of residual spermatogenesis in non-obstructive azoospermic men. Hum Reprod 2014; 29(12): 2620-7.
  20. Fischer AH, Jacobson KA, Rose J, Zeller R. Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc 2008; 2008: db.
  21. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc 2006; 1(3): 1559-82.
  22. Tateishi K, Okada Y, Kallin EM, Zhang Y. Role of Jhdm2a in regulating metabolic gene expression and obesity resistance. Nature 2009; 458(7239): 757-61.
  23. Kouadjo KE, Nishida Y, Cadrin-Girard JF, Yoshioka M, St-Amand J. Housekeeping and tissue-specific genes in mouse tissues. BMC Genomics 2007; 8: 127.
  24. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett 2004; 26(6): 509-15.
  25. Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 2004; 15(3): 155-66.
  26. Karge WH, III, Schaefer EJ, Ordovas JM. Quantification of mRNA by polymerase chain reaction (PCR) using an internal standard and a nonradioactive detection method. Methods Mol Biol 1998; 110: 43-61.
  27. Brunner AM, Yakovlev IA, Strauss SH. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 2004; 4: 14.
  28. Ji H, Wang J, Liu J, Guo J, Wang Z, Zhang X, et al. Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (Anser anser domestica) Gene Expression. Asian-Australas J Anim Sci 2013; 26(3): 423-32.
  29. Zhou YH, Raj VR, Siegel E, Yu L. Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes. Biomark Insights 2010; 5: 79-85.
  30. Ferguson BS, Nam H, Hopkins RG, Morrison RF. Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes. PLoS One 2010; 5(12): e15208.
  31. Fernandes JM, Mommens M, Hagen O, Babiak I, Solberg C. Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development. Comp Biochem Physiol B Biochem Mol Biol 2008; 150(1): 23-32.
  32. Spinsanti G, Panti C, Lazzeri E, Marsili L, Casini S, Frati F, et al. Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies. BMC Mol Biol 2006; 7: 32.
  33. Zornhagen KW, Kristensen AT, Hansen AE, Oxboel J, Kjaer A. Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction. Vet Comp Oncol 2015; 13(4): 485-93.
  34. Rocha DJ, Santos CS, Pacheco LG. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis. Antonie Van Leeuwenhoek 2015; 108(3): 685-93.
  35. Nadai C, Campanaro S, Giacomini A, Corich V. Selection and validation of reference genes for quantitative real-time PCR studies during Saccharomyces cerevisiae alcoholic fermentation in the presence of sulfite. Int J Food Microbiol 2015; 215: 49-56.
  36. Liu Z, Zhu Q, Li J, Yu J, Li Y, Huang X, et al. Selection and evaluation of reference genes for expression analysis of Cassi. Biosci Biotechnol Biochem 2015; 79(11): 1818-26.
  37. Iser IC, de Campos RP, Bertoni AP, Wink MR. Identification of valid endogenous control genes for determining gene expression in C6 glioma cell line treated with conditioned medium from adipose-derived stem cell. Biomed Pharmacother 2015; 75: 75-82.
  38. Ayakannu T, Taylor AH, Willets JM, Brown L, Lambert DG, McDonald J, et al. Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma. Mol Hum Reprod 2015; 21(9): 723-35.
مجله دانشکده پزشکی اصفهان
دوره 33، شماره 367 - شماره پیاپی 367
بهمن و اسفند 1394
صفحه 2407-2416

فایل ها

سابقه مقاله

  • تاریخ دریافت: 20 مهر 1394
  • تاریخ بازنگری: 11 آبان 1403
  • تاریخ پذیرش: 17 فروردین 1401

هم رسانی

ارجاع به این مقاله

آمار