A Single-Tube Multiplex Qpcr Assay for Mitochondrial DNA (Mtdna)
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Turk J Biochem 2019; 44(6): 769–777 Research Article Hasan Basri Kiliç, Bengisu Kevser Bulduk and Y. Çetin Kocaefe* A single-tube multiplex qPCR assay for mitochondrial DNA (mtDNA) copy number assessment Mitokondriyal DNA (mtDNA) Kopya Sayısı Belirlemede Tek Tüpte Multiplex qPCR Yöntemi https://doi.org/10.1515/tjb-2018-0372 quantification of mtDNA copy number. Our findings Received September 4, 2018; accepted November 16, 2018; previ- show higher accuracy and reproducibility over canonical ously published online December 25, 2018 approach, reducing cost and error rate. Abstract Keywords: Mitochondrial DNA copy number; Multiplex Objective: Detection of mtDNA copy number is required real-time quantitative PCR; Molecular beacon probes; for diagnosis of mtDNA depletion. Multiplex quantifica- Mitochondrial depletion. tion of mtDNA in blood samples was claimed via normal- izing to a nuclear single copy gene using qPCR. This is not Öz possible in high mtDNA samples due to template abun- dance. Multiplex qPCR assays cannot be normalized to Amaç: Mitokondriyal deplesyon tanısında, mtDNA kopya single copy sequences of the nuclear genome. sayısının belirlenmesi gerekmektedir. Bu amaçla qPCR Methods: mtDNA quantification was tested normalizing tekniği ile mtDNA sayısı, genomda yer alan tek kopya dizi- to a single copy nuclear gene via singleplex and multi- lere oranlanmaktadır. Multipleks qPCR ile kan dokusunda plex reactions. Failure in normalization directed to design gerçekleştirilebilen nicel ölçüm, hedef DNA kalıbı oranın- and test targeting multi-copy 18S rDNA gene with success. daki dengesizlik nedeni yüksek mtDNA içeren dokularda mtDNA quantification was standardized both in separate uygulanamamaktadır. and multiplexed single-tube reactions based on molecular Gereç ve Yöntem: Nicel mtDNA kopya sayısı çekirdek geno- beacon technology. munda yer alan tek kopya diziye oranlanarak belirlen- Results: mtDNA copy number assessment cannot be nor- meye çalışıldı. Farklı tüplerdeki tepkimelerde başarılı olan malized to a single copy sequence in high-copy-number işlemin mültipleks tepkimede başarısız olduğu gözlendi. tissues. However, normalizing mtDNA to the nuclear 18S Bireyler arasında tekrar sayısı değişmeyen, çok kopya bir rDNA multiple copy sequence is amenable to be standard- referans dizi olarak 18S rDNA genini hedefleyen bir qPCR ized in single tube. When compared, multiplexing exhib- yaklaşımı tasarlandı ve denendi. Moleküler beacon tekno- ited higher resolution power for quantification of mtDNA in lojisi kullanımı ile tasarlanan qPCR tepkimesi ayrı tüplerde various samples from the most abundant to the scant ones. ve tek tüpte (mültipleks) standardize edildi. Conclusion: We describe a multiplex assay that can Bulgular: Yüksek kopya mtDNA içeren dokularda, mtDNA be translated as a standard technique for single-tube sayısı, çekirdek genomunda yer alan tek kopya dizi refe- rans alınarak tek tüpte belirlenemedi. Bu durumun nedeni- *Corresponding author: Y. Çetin Kocaefe, MD, PhD, Hacettepe nin, iki amplikon arasındaki 8 döngü fark olduğu anlaşıldı. University School of Medicine, Department of Medical Biology, Oranlama için genomda çok kopya halinde bulunan 18S Ankara, Turkey, Phone: +90 312 3052541, Fax: +90 312 3096060, rDNA dizisi kullanılarak, aynı tepkime tüpü içinde mtDNA e-mail: [email protected]. kopya sayısı tekrarlanabilir ve güvenilir şekilde belirlendi. https://orcid.org/0000-0003-3216-9399 Sonuç: mtDNA kopya sayısı belirlemede, etkin ve güvenilir Hasan Basri Kiliç and Bengisu Kevser Bulduk: Hacettepe University School of Medicine, Department of Medical Biology, Ankara, bir standart teknik olarak kullanılmak üzere tek-tüp (mül- Turkey, e-mail: [email protected] (H. B. Kiliç); tipleks) qPCR testi geliştirilmiş ve standardize edilmiştir. [email protected] (B. K. Bulduk) Tanımlanan test, güvenilirliği ve tekrarlanabilirliğinin 770 Hasan Basri Kiliç et al.: A single-tube multiplex qPCR assay for mitochondrial DNA yanında maliyet ve hata risklerini azaltarak farklı tüplerde number in blood tissue is misleading when mtDNA deple- gerçekleştirilen qPCR yaklaşımına üstünlük sağlamaktadır. tion is a suspected clinical diagnosis. Quantitative PCR is the most widely used technique Anahtar kelimeler: Mitokondriyal DNA kopya sayısı; for determining mtDNA copy number. There is a require- Mitokondriyal deplesyon; Genetik tanı; Polimeraz zincir ment for absolute quantitation of viral genome assess- tepkimesi. ment in microbiology or detection of mutation load in oncology. However, in the case of mtDNA copy number analysis, a relative quantitative comparison to control Introduction samples is accepted to be adequate. The assessment of mtDNA content in DNA samples is achieved by normali- Mitochondrion is the essential organelle of eukaryotic evo- zation of an mtDNA amplicon to the nuclear genome lution exhibiting a conserved set of homologous genetic that serves as a reference. Thus, mtDNA quantification material throughout all eukaryotic lineages [1]. In humans, requires assessment of both mtDNA and genomic copy maternally inherited mitochondrial genome (mtDNA) is a numbers. Relative quantification of mtDNA was previ- circular, 16.569 base-pair-long extra-chromosomal DNA ously investigated by others using canonical qPCR assay, molecule that harbours 37 genes, encoding 13 proteins to targeting the amplification of mitochondrial and genomic contribute to the subunits of oxidative phosphorylation DNA in separate tubes to provide a relative copy number complexes excluding complex II. Twenty-four remaining [13–17]. Comparison of two amplicons in separate tubes is genes transcribe two rRNA and 22 tRNA molecules to con- open to technical pipetting errors due to the nature of the tribute to the mitochondrial protein synthesis [2]. Replica- PCR reaction or DNA quality. To overcome these poten- tion and transcription of mtDNA are strongly coupled [3]. tial errors, multiplexing of nuclear and mtDNA ampli- The enzymes required for the synthesis and maintenance cons were also tested in a single-tube qPCR approach of mtDNA are encoded by the nuclear genome including [18]. While this study aimed the assessment of relative mtDNA specific DNA polymerase POLG1. Mutations in mtDNA copy number in blood samples, mtDNA content the nuclear genes that encode these proteins result in a in white blood cells are much lower than high-energy- distinct subtype of mitochondrial cytopathy disorders demand tissues. However, multiplexing of two separate exhibiting Mendelian inheritance and a phenotype simi- amplicons in a single tube is strictly limited by the avail- larity due to diminished mtDNA content named as “mito- ability of target sequences. In this latter case, the mtDNA chondrial depletion” [4]. Major nuclear genes required copy number in high-energy-demand tissues is much for the maintenance of mtDNA and responsible for such higher than a single-gene sequence of nuclear genome rare Mendelian disorders are summarized in Table 1. Due [12]. Abundant mtDNA amplicon is expected to be ampli- to the high-consanguinity rate, such rare disorders are fied (and reach PCR plateau) much before a competing encountered at a much higher frequency in Turkish popu- single-copy genomic amplicon, completely obscuring the lation [11]. Tissues with higher energy demand likewise amplification of the latter. Thus, availability of a nuclear- the skeletal muscle, heart, kidney, liver and brain are pri- genome normalizer sequence is the limiting factor for marily affected by the structural and functional defects of successful multiplexing of the two amplifications in a mtDNA. On the other hand, white blood cells with rela- single tube. From this point forth, here we describe and tively lower energy demand and number of mtDNA are the validate a single-tube quantitative PCR approach based least impaired under depletion conditions compared to on molecular beacon technology which is amenable for the above tissues [12]. Thus, assessment of mtDNA copy optimization as a routine single-tube assay. Table 1: Major nuclear genes responsible for the maintenance of mtDNA and related disorders. Gene/Enzyme Clinical course Symptoms Diagnosis TK2/Thymidine kinase 2 Myopathic depletion syndrome [5] Weakness of muscles [6] High creatine kinase [7] DGUOK/ Mitochondrial hepatoencephalopathy, hepatic Liver impairment [6] Deoxyguanosine kinase and neurologic symptoms during infancy [8] POLG1/DNA polymerase Encephalopathy, hepatic impairment, Seizures, ataxia, gamma epilepsy, ataxia, neuropathy [9] polyneuropathy [6] TYMP/Thymidine Mitochondrial neurogastrointestinal Gastrointestinal symptoms, High thymidine [7] phosphorylase encephalomyopathy (MNGIE) [10] anorexia, polyneuropathy [6] Hasan Basri Kiliç et al.: A single-tube multiplex qPCR assay for mitochondrial DNA 771 Materials and methods observe the outcome of multiplexing. The reaction was setup in 0.1 mL thin-walled PCR tubes at 10 μL volume containing 2 μL of GoTaq 5X PCR Buffer (Promega), sup- Patients and samples plemented with 3.5 mM MgCl2, 0.4 mM dNTPs (Promega), 4 pMole of both forward and reverse primers, 0.3 mM Peripheral blood samples and muscle biopsies from pae- SYTO9 dye (Invitrogen), 0.1 U of GoTaq DNA polymerase diatric-age patients with suspected mtDNA depletion syn- (5 U/μL) and 100 mMole of DNA template. Multiplex reac- dromes or mitochondrial cytopathies that were referred tions were employed to include two sets of primers sup- from the paediatric neurology and metabolism units of plied at the above