Özgecan KORKMAZ AĞAOĞLU, Ali Osman TURGUT

Kantitatif RT-PCR (RT-qPCR) Uygulamalarında Kullanılan RNA’ların Kalite Kontrolleri ve Önemi

RNA kalitesi, RT-qPCR performansı ve güvenilirliği açısından dikkat edilmesi gereken önemli bir husustur. RT-qPCR çalışmalarında RNA kalite değerlendirilmesine dikkat edilmemesi, yapılan bilimsel çalışmaların güvenilirliği açı-sından önemli bir risk oluşturmaktadır. Bu noktada, RT-qPCR çalışmasının her aşamasında gerekli standartlara uyul-ması ve RNA kalite değerlendirmesinin mutlaka yapılması gerekmektedir. RNA kalitesinin çalışmanın her aşamasında yüksek tutulması, RT-qPCR verimliliğinin ve elde edilen sonuçların güvenilirliğinin artırılmasına olanak tanımaktadır. Yapılan çok sayıda çalışma RNA kalitesinin RT-qPCR sonuçları üzerine olan etkisini ve RNA kalite değerlendirmesinin gerekliliğini ortaya koymaktadır. Bu derlemede, RNA kalitesi, RNA kalite değerlendirmesine kullanılan yöntemler ve RNA kalitesinin RT-qPCR performansı üzerine etkilerinden bahsedilmiştir.

Anahtar Kelimeler:

Gen ekspresyonu, MIQE, RNA kalitesi, RT-qPCR

Quality Controls and Importance of RNAs Used in Quantitative RT-PCR (RT-qPCR) Applications

RNA quality is an important consideration for RT-qPCR performance and reliability. The lack of attention to RNA quality assessment poses a significant risk for the reliability of scientific studies in RT-qPCR studies. At this point, the required standards must be followed, and RNA quality assessment must be performed at every stage of the RT-qPCR study. Maintaining high RNA quality at each stage of the study enables to increase efficiency of RT-qPCR and reliability of the results. Numerous studies have demonstrated the effect of RNA quality on RT-qPCR results and the need for RNA quality assessment. In this review; RNA quality, methods used for RNA quality assessment and the ef-fects of RNA quality on RT-qPCR performance are discussed.

Keywords:

Gene expression, MIQE, RNA quality, RT-qPCR,

PDF

___

Agaoglu OK, Agaoglu AR, Guzeloglu A, Aslan S, Kurar E, Kayis SA, Schäfer-Somi S. Gene expres-sion profiles of some cytokines, growth factors, re-ceptors and enzymes (GM-CSF, IFNγ, MMP-2, IGF-II, EGF, TGF-β, IGF-IIR) during pregnancy in the cat uterus. Theriogenology 2016; 85(4): 638-44.
Agaoglu OK, Agaoglu AR, Guzeloglu A, Kurar E, Kayis SA, Ozmen O, Aslan S. Expression of hypox-ia inducible factors and vascular endothelial growth factor during pregnancy in the feline uterus. Theri-ogenology 2015; 84(1): 24-33.
Becker C, Hammerle-Fickinger A, Riedmaier I, Pfaffl MW. mRNA and MicroRNA quality control for RT-qPCR analysis. Methods 2010; 50(4): 237-43.
Bustin SA, Benes V, Garson J, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guide-lines: minimum information for publication of quanti-tative real-time PCR experiments. Clin Chem 2009; 55: 611-22.
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162(1): 156-9.
Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction: twenty-something years on. Nature Protocols 2006; 1(2): 581-5.
Das PJ, Pari N, Gustafson-Seabury A, Vishnoi M, Chaki SP, Love CC, Varner DD, Chowdhary BP, Raudsepp T. Total RNA isolation from stallion sperm and testis biopsies. Theriogenology 2010; 74(6): 1099-106.
Die JV, Román B. RNA Quality assessment: A view induces from plant qPCR Studies. J Exp Bot 2012; 63(17): 6069-77.
Esser KH, Marx WH, Lisowsky T. Nucleic acid free matrix: regeneration of DNA binding columns. Biotechniques 2005; 39(2): 270-1.
Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 2006; 27(2-3): 126-39.
Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl MW. Comparison of relative mRNA quantification models and the impact of RNA integrity in quantita-tive real-time RT-PCR. Biotechnol Lett 2006; 28: 1601-13.
Gjerse DT, Hoang L, Hornby D. RNA Purification and Analysis: Sample Preparation, Extraction, Chroma-tography. First Edition. Weinheim, Germany: Wiley-VCH, 2009; p. 4.
Green MR, Sambrook J. Quantification of RNA by Real-Time reverse transcription-polymerase chain reaction (RT-PCR). Cold Spring Harb Protoc 2018; 10: 847-56.
Hammerle-Fickinger A, Riedmaier I, Becker C, Meyer HHD, Pfaffl MW, Ulbrich SE. Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression anal-yses. Biotechnol Lett 2009; 32: 35-44.
Huang Q, Fu WL. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clin Chem Lab Med 2005; 43(8): 841-2.
Imbeaud S, Graudens E, Boulanger V, Barlet X, Za-borski P, Eveno E, Mueller O, Schroeder A, Auffray C. Towards standardization of RNA quality assess-ment using user-independent classifiers of microca-pillary electrophoresis traces. Nucleic Acids Res 2005; 33(6): 1-12.
Ingham DJ. The study of transgene copy number and organization. Methods Mol Biol 2005; 286: 273-90.
Johnson VJ, Yucesoy B, Luster MI. Genotyping of single nucleotide polymorphisms in cytokine genes using real-time PCR allelic discrimination technolo-gy. Cytokine 2004; 27: 135-41.
Karlsson O, Segerström L, Sjöback R, Nylander I, Boren Mats. QPCR Based mRNA quality score show intact mRNA after heat stabilization. Biomol Detect Quantif 2016; 7: 21-6.
Kojima K, Ozawa S. Method for isolating and purify-ing nucleic acids. United State patent 2002; Patent no: US 6,905,825 B2.
Kozera B, Rapacz M. Reference genes in Real-Time PCR. J Appl Genetics 2013; 54: 391-406.
Li Y, Wang K, Chen L, Zhu X, Zhou J. Quantification of mRNA levels using Real-Time Polymerase Chain Reaction (PCR). Methods Mol Biol 2016; 1406: 73-9.
McGovern F, Tommy B, Marion R, Torres S. Assess-ment of RNA stability in postmortem tissue from New-Born lambs. Anim Biotechnol 2018; 29(4): 269-75.
Nielsen H. Working with RNA. Nielsen H eds. Methods in Molecular Biology. 2010; pp. 15-28.
Nilsen TW. The fundamentals of RNA purification. Cold Spring Harb Protoc 2013; 7: 618-24.
Nolan T, Bustin SA. Procedures for quality control of RNA samples for use in quantitative reverse tran-scription PCR. Keer JT, Birch L. eds. In: Essentials of Nucleic Acid Analysis: A Robust Approach, 2008; pp.189-207.
Nolan T, Hands RE, Bustin SA. Quantification of mRNA using Real-Time RT-PCR. Nat Protoc 2006; 1(3): 1559-82.
Opitz L, Salinas-Riester G, Grade M, Jung K, Jo P, Emons G, Ghadimi BM, Beissbarth T, Gaedcke J. Impact of RNA degradation on gene expression profiling. BMC Med Genet 2010; 3 (36): 1-14.
Özalp GR, Şimşek G, Akçağlar S, Shenavai S. Trizol RNA Ekstraksiyon Metodu: İnek plasentomu için oldukça etkili metod değerlendirmesi. Uludag Univ J Fac Vet Med 2010; 29(1): 1-6.
Pazzagli M, Malentacchi F, Simi L, Orlando C, Wyrich R, Günther K, Hartmann CC, Verderio P, Pizzamiglio S, Ciniselli CM, Tichopad A, Kubista M, Gelmini S. SPIDIA-RNA: first external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses. Methods 2013; 59(1): 20-31.
Pérez-Novo CA, Claeys C, Speleman F, Van Cau-wenberge P, Bachert C, Vandesompele J. Impact of RNA quality on reference gene expression stability. BioTechniques 2005; 39: 52-56.
Riedmaier I, Bergmaier M, Pfaffl MW. Comparison of two available platforms for determination of RNA quality. Biotechnol Biotec Eq 2011; 24: 2154-9.
Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Ethanol precipitation of RNA and the use of carriers. Cold Spring Harb Protoc 2010b; 6: 1-5.
Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Guide-lines for the use of RNA purification kits. Cold Spring Harb Protoc 2010a; 7: 1-4.
Romero IG, Pai AA, Tung J, Gilad Y. RNA-seq: im-pact of RNA degradation on transcript quantifica-tion. BMC Biol 2014; 12(42): 1-13.
Sambrook J, Russel D. Molecular Cloning: A Labora-tory Manual. Third Edition. New York, USA: Cold Spring Harbor Laboratory Press, 2001; p. 613.
Schoor O, Weinschenk T, Hennenlotter J, Corvin S, Stenzl A, Rammensee H-G, Stevanović S. Moder-ate degradation does not preclude microarray anal-ysis of small amounts of RNA. Biotechniques 2003; 35: 1192-201.
Sethi N, Palefsky J. Transcriptional profiling of dys-plastic lesions in K14-HPV16 transgenic mice using laser microdissection. FASEB J 2004; 18(1): 1243-5.
Tan SC, Yiap BC. DNA, RNA, and protein extraction: The past and the present. J Biomed Biotechnol 2009; 2009: 1-10.
Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. A Practical approach to RT-qPCR-Publishing data that conform to the MIQE quidelines. Methods 2010; 50(4): 1-5.
Ullrich A, Shine J, Chirgwin J, Pictet R, Tischer E, Rutter WJ, Goodman HM. Rat insulin genes: Con-struction of plasmids containing the coding se-quences. Science 1977; 196(4296): 1313-9. Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ 2005; 29(3): 151-9.
VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 2008; 44(5): 619-26.
Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S, Hellemans J, Speleman F, Vandesompele J. Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res 2011; 39(9): e63.
Walker SE, Lorsch J. RNA Purification - Precipitation Methods. Methods Enzymol 2013; 530: 337-43.