A Single-Tube Multiplex Qpcr Assay for Mitochondrial DNA (Mtdna)

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 normalizing to a nuclear single copy gene using qPCR. This is not Öz possible in high mtDNA samples due to template abundance. 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 referans 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 Sonuc: 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 concentrations. Reaction conditions the İhsan Doğramacı Children’s hospital for diagnostic were initial denaturation of 2 min at 94°C, followed by 40 purposes. All participants’ rights were protected and a cycles of 5 s at 94°C and 15 s at 60°C. A Corbett Rotorgene written informed consents were obtained before the pro- 6000 thermal cycler was used and fluorescent signal cedures according to the Helsinki Declaration. The study acquisition was achieved at the end of each elongation protocol and the written extended consent was approved step. Four logs of serial dilutions of a pool of control skel- by the local Ethics Committee of Hacettepe University (GO etal muscle DNA samples were used to calculate a stand- 17/519-08). All blood samples were processed within 48 h ard curve. All reactions were triplicated and the average of sampling and muscle biopsies were kept at −80°C until fluorescent signal intensity of triplicates was used for DNA extraction using standard technique [8]. PCR amplification curves using Rotor-Gene Q software (Version 1.7, Build 94). 2-∆∆CT method was used for quantitative assessment and normalization of samples. Fur- Quantitative real-time PCR (qRT-PCR) thermore, PCR reactions were paused at cycles 15, 22, 29 and 36 and products were run on agarose gel to achieve a Two separate approaches were conducted for the assess- visual semi-quantitative assessment. This approach also ment of mtDNA copy number in DNA samples. First, a confirmed the absence of non-specific products, primer- SYBRGreen intercalating dye approach was pursued for dimers or smears that may interfere with intercalating the relative assessment of mtDNA content in separate fluorescence. tubes. Strict compliance to guidelines was followed for A second PCR reaction was designed to amplify and the primer design and standardization of qPCR assays quantitate a dissimilar 148 bp fragment of MT-CYB gene. using intercalating dyes [19]. A 76 bp fragment of the This second amplicon was normalized to a 120 bp ampli- mtDNA targeting mitochondrial cytochrome B gene (MT- con targeting the multiple-copy 18S rDNA nuclear gene CYB) was targeted and normalized to a 147 bp amplicon (RNR). A similar PCR setup likewise the above mentioned targeting the nuclear, single copy albumin gene (ALB) protocol was employed and a semi-quantitative assess- sequence. All primer and probe sequences are provided ment was done on agarose gels on 15, 22, 29 and 36 cycles. in Table 2. The absence of non-specific products, primer-dimers or PCR reactions were carried out in either separate smears was also evaluated. tubes as singleplex reactions or were multiplexed to Single-tube multiplex qPCR amplification and fluoro- scopic detection of the two above targets were achieved Table 2: Primer and probe sequences used in the study. using molecular beacon technology. Two molecular beacon probes targeting the MT-CYB and RNR amplicons Gene Primers and probes were designed to provide 5′ end Cy5 emission quenched Albumin (ALB) 5′-CCAAAGTCCACACGGAATGC-3′ by 3′ end Black Hole Quencher 2 (BHQ2) and 5′ end Hex Intercalating 5′-CATTGGTGATAGCTGACAGTGG-3′ emission quenched by Black Hole Quencher 1 (BHQ1), Cytochrome B (MT-CYB) 5′-TTCGCCCACTAAGCCAATCAC-3 respectively (Metabion Int.). Thermocycler conditions Intercalating 5′-CTAACCTGAATCGGAGGACAACC-3′ were modified to provide an initial denaturation of ′ ′ 18 S rDNA (18S) 5 -GGTTGATCCTGCCAGTAGC-3 94°C for 2 min followed by 40 cycles of 5 s at 94°C, 20 s Beacon 5′-GGTTCCTTTGGTCGCTCG-3′ ° ° 5′ (Hex)-CGCGATCTGCATGTCTGAGTAC at 52 C and 15 s at 72 C. Probes were supplemented at a GCACGGCGATCGCG-(BHQ1)3′ final concentration of 0.4 mM in each PCR reaction. Fluo- Cytochrome B (MT-CYB) 5′-CGACCCAGACAATTATACC-3′ rescent signal acquisitions were done at the end of each Beacon 5′-GATGAGGATGGATAGTAATAGG-3′ annealing step at channels red (625 nm emission, 660 nm 5′ (Cy5)-CGCGATCTTCCTATTCGCCTAC acquisition for Cy5) and yellow (530 nm emission, 550 nm ACAATTCTCCGGATCGCG-(BHQ2)-3′ acquisition for Hex). 772 Hasan Basri Kiliç et al.: A single-tube multiplex qPCR assay for mitochondrial DNA

Statistics (75 bases) was amplified and normalized to the relative copy number of a 147 bp nuclear genome fragment of a The relative mtDNA copy number variations between single copy sequence of ALB gene using SybrGreen tech- control and mtDNA depleted patient samples were tested nique. A pool of control skeletal muscle DNA samples was for significance using one way ANOVA test. The consist- setup to amplify the target sequences in separate tubes ency of singleplex and multiplex reactions were confirmed in triplicates and a relative quantitation was assessed by correlation analysis. Statistical modules of Microsoft using delta-delta Ct method (2-∆∆CT) as described previ- Excel (2010) were employed. ously [20]. Results showed consistent relative mtDNA copy number in standards encompassing four logs of serial dilutions (R2 > 0.99). The fluorescent signal emission of MT-CYB amplicons reached the same intensity Results of the ALB gene 8( ± 0.4) cycles before regardless of the starting DNA amount (Figure 1). When skeletal muscle In order to demonstrate the technical limits of “single tube DNA samples from the individuals that consisted the multiplex” quantitation of mtDNA using real-time PCR, pool of controls were individually assessed, this ~8-cycle- initially we performed a canonical approach. A sequence difference was observed to be consistent throughout the of mtDNA genome corresponding to the MT-CYB gene samples (Figure 2A and B). The average Ct values were

A MT-CYB ALB

0.1

ed· fluorescence (log) 0.01 maliz MT-CYB ALB

Nor R = 0.99898 R = 0.99846 R2 = 0.99796 R2 = 0.99693 M = –3.326 M = –3.371 B = 26.374 B = 37.322 Eff. = 1.00 Eff. = 0.98 0.00 5 10 15 20 25 30 35 40 16.4 Ct B Ct

25 1.5

16.4 15

0 MTCYB MTCYB–1 MTCYB–2 MTCYB–3 ALB ALB–1 ALB–2 ALB–3 9.7 13.1 16.4 19.7 17.9 20.8 24.0 27.8

Figure 1: A sequence of mtDNA (MT-CYB gene in red) was amplified and normalized to the relative copy number of a single copy nuclear sequence (ALB gene in blue) using SybrGreen technique in triplicates and separate tubes. A set of control skeletal muscle DNA samples were pooled to amplify the target sequences and real-time amplification curves obtained from four-log serial dilution standards are plotted (A – Dotted lines show average of triplicates). Average Ct values of target amplicons for each dilution are shown in bar graph (B – MT-CYB gene in red and ALB gene in blue). MT-CYB amplicon for the third log dilution exhibited a Ct value of 16.4 where undiluted ALB amplicon exhibited the same fluorescence at Ct = 17.9 exhibiting a 1.5 cycle difference (see discussion). Hasan Basri Kiliç et al.: A single-tube multiplex qPCR assay for mitochondrial DNA 773

A MT-CYB ALB

0.1 ed· fluorescence (log) 0.01 maliz MT-CYB ALB Nor R = 0.99898 R = 0.99846 R2 = 0.99796 R2 = 0.99693 M = –3.326 M = –3.371 B = 26.374 B = 37.322 Eff. = 1.00 Eff. = 0.98 0.00 5 10 15 20 25 30 35 12.1 20.5 B Ct 25

20.5 20

12.1 10

0 C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C6

MT-CYB ALB

ALB MT-CYB ALB + MT-CYB C d d d –1 –2 –3 –1 –2 –3 –1 –2 –3 C1 C1 C1 C1 Deplete C2 C1 C1 C1 C1 Deplete C2 C1 C1 C1 C1 Deplete C2

200 bp Cycle 15 100 bp

200 bp Cycle 22 100 bp

200 bp Cycle 29 100 bp

200 bp Cycle 36 100 bp

Figure 2: MT-CYB and ALB fragments were amplified using individual skeletal muscle DNA samples that consisted the pool of controls. Amplification curves are plotted in (A) and shown in bar graph in (B) (MT-CYB in red and ALB in blue). The average Ct values are 12.1( ± 1.19) for MT-CYB and 20.5( ± 0.28) for ALB showing 8.4 cycle Ct difference. PCR reactions amplifying MT-CYB and ALB fragments (singleplex/separate tubes and multiplex/single tube) were performed using the control pool, depleted mtDNA sample and a single control. The PCR reactions were interrupted in the linear amplification phase at cycles 15, 22, 29 and 36 and the products are visualized on 2.5% agarose gel (C – singleplex ALB amplicon on the left, singleplex MT-CYB in the middle and multiplex reaction on the right). 147 bp ALB amplicon product was not visible in multiplex reactions. 774 Hasan Basri Kiliç et al.: A single-tube multiplex qPCR assay for mitochondrial DNA

18S MT-CYB 18S + MT-CYB A d d d –1 –2 –3 –1 –2 –3 –1 –2 –3 C1 C1 C1 C1 Deplete C2 C1 C1 C1 C1 Deplete C2 C1 C1 C1 C1 Deplete C2 200 bp Cycle 15 100 bp

200 bp

Cycle 22 100 bp

200 bp

Cycle 29

100 bp

200 bp

Cycle 36

100 bp B

0.1

ed· fluorescence (log) 0.01

aliz Singleplex MT-CYB 18S No rm R = 0.99935 R = 0.99746 R2 = 0.99871R2 = 0.99493 M = –3.692M = –3.572 B = 29.594 B = 37.322 Eff. = 0.87 Eff. = 0.91 0.00 5 10 15 20 25 30 35 C

0.1

0.01

ed· fluorescence (log) Multiplex aliz MT-CYB 18S R = 0.99945 R = 0.99641 No rm R2 = 0.99291 R2 = 0.99283 M = –3.331 M = –3.315 B = 25.273 B = 34.518 Eff. = 1.00 Eff. = 1.00 0.00 5101520253035 D 160

140

120 e

100

e mtDNA percentag 60

Relativ 40 x

20 x Singleple 0 Multiple Control 1 Control 2Control 3mt.3290T>C Common dGUOK TK2 TYMP deletion Multiplex 115.88 143.36 40.76 2.45 6.0320.87 0.04 10.96 Singleplex 61.28 117.40 40.76 0.49 0.99 3.71 0.01 4.69 Hasan Basri Kiliç et al.: A single-tube multiplex qPCR assay for mitochondrial DNA 775

Figure 3: PCR reactions amplifying MT-CYB and 18S rDNA fragments (singleplex/separate tubes and multiplex/single tube) were performed using the control pool, depleted mtDNA sample and a single control. The PCR reactions were interrupted in the linear amplification phase at cycles 15, 22, 29 and 36 and the products are visualized on 2.5% agarose gel (A – singleplex 18S rDNA amplicon on the left, singleplex MT-CYB in the middle and multiplex reaction on the right). 18S rDNA amplicon was visible alongside MT-CYB amplicon in multiplex reactions in cycles 29 and onward for control samples (A, right panel). Depleted mtDNA sample revealed 18S rDNA amplicon at cycle 22 proceeding MT-CYB. Singleplex and multiplex reactions were carried out in qPCR using molecular beacon probes and logarithmic amplification curves are plotted (B – singleplex amplification curves in separate tubes and C – multiplex amplification curves in a single tube. MT-CYB is in red and 18S rDNA is in green). Relative quantitation was assessed for selected samples including controls of variant mtDNA content alongside samples with various mitochondrial cytopathies including depletion syndromes. Relative mtDNA content of the samples normalized to the average of the controls are plotted in bar graph (D).

12.1( ± 1.19) and 20.5( ± 0.28) for MT-CYB and ALB, respec- in both singleplex and multiplex reactions (Figure 3A). tively showing 8.4 cycle difference. The linear amplifica- In contrast to the results obtained with the ALB gene, tion phase of MT-CYB amplicons were between cycles 11 the multi-copy template was co-amplified alongside the and 15. This is much before the initial detection of any of mtDNA template from cycle 22 and onward. Furthermore, the ALB gene amplicons (cycles 16–17). both amplicons were visible in multiplexed reactions in Based on these results we postulate that the outnum- cycle 29 onward. In multiplex reactions, 18S amplicon bered abundance of mtDNA template results in rapid was detected earlier in a DNA sample harbouring mtDNA draining of PCR substrates in early cycles, eliminating depletion. These results suggested that 18S rDNA sequence amplification possibility of any low-abundant single copy to be a valid control for the standardization of single-tube reference template in the same reaction tube. In order to multiplex mtDNA quantitation. verify this hypothesis, we multiplexed mtDNA and nuclear In the light of above data, we designed molecular templates in a single-tube to amplify concomitantly. The beacon probes targeting MT-CYB and 18S rDNA sequences PCR reaction was interrupted in the linear amplification to quantify these amplicons multiplexed in a single tube. phase at cycles 15, 22, 29 and 36. The abundances of prod- We sought to validate the performance of the beacon ucts were visualized on agarose gel. MT-CYB amplicon was probes by testing the same sample set in separate tubes as visible in both singleplex and multiplexed reactions start- well as a multiplexed single tube PCR reaction (Figure 3B ing from the 15th cycle onward. However, ALB gene PCR and C, respectively). Amplification of targets in separate product was only detectable following cycle 29 in singleplex tubes caused altered but acceptable variation. Quanti- reactions while it was not visible in any of the multiplexed fication of DNA samples from individuals with various reactions (Figure 2C). This observation supported the above mitochondrial cytopathies revealed consistent results in hypothesis. Furthermore, a DNA sample exhibiting mtDNA both approaches (Figure 3D). Relative mtDNA copy ratio depletion (%5) was also included in this experiment. The of all mitochondrial cytopathy patients was lower than mtDNA amplicon of the depleted sample was visible in 20% of the average of control samples exhibiting extreme cycle 22 (compared to cycle 15 for the controls), however, variation. The lowest mtDNA content was observed in an the PCR product of the nuclear genome was not visible by individual with thymidine kinase 2 (TK2) mutation. Two the end of the cycle 36 in the multiplexed reaction. experimental approaches were compared for consist- Above results pinpointed the fact that the discrep- ency and the significance of mtDNA copy number varia- ancy of template abundance was the major obstacle for tion between controls versus mtDNA cytopathy samples concomitant amplification of both products in a single were tested with ANOVA. This comparison revealed tube. In order to circumvent this template abundance dif- highly similar p values of 0.0070 and 0.0054 in singleplex ference, we aimed to test 18S rRNA multi-copy gene as the and multiplex reactions, respectively. Furthermore, the reference template for nuclear DNA. A similar experimen- equivalency of these techniques was validated by corre- tal approach was conducted to amplify mtDNA (MT-CYB) lation analysis that revealed a correlation coefficiency of and 18S ribosomal RNA (RNR) gene fragment present in all R2 = 0.96 for singleplex or multiplex quantification. RNR gene clusters (RNR1 to RNR5) [21]. Same experimental setup was replicated using 18SRNR template replacing ALB gene. Singleplex and multiplex PCR reactions were Discussion interrupted in various cycles as above and analysed on agarose gel. Results showed that the amplicon targeting PCR technologies are widely used for the assessment the mtDNA was visible starting from cycle 15 and onward of target DNA samples in various specimens. The wide 776 Hasan Basri Kiliç et al.: A single-tube multiplex qPCR assay for mitochondrial DNA application spectra of this approach include environ- In order to circumvent this problem of imbalanced mental health, microbiology and clinical oncology. The template abundance, we suggest using a stable multi-copy above fields require the absolute quantitation of a target sequence residing within the nuclear genome. 18S rDNA sequence since the abundance of a rare target is critical. In harbours such properties to serve as a reliable normal- the case of mitochondrial cytopathies, a subset of condi- izer template with its presence in five clusters of 200–300 tions impairs the replication and maintenance of mtDNA total copies in haploid genome and not exhibiting inter- that it’s copy number is depleted in effected tissues. In sus- individual variance [21]. When multiplex qPCR approach pected cases, a relative comparison of mtDNA copy number was applied on 18S and MT-CYB, successful amplification is required to support diagnosis before any further directed could be achieved in multiplex reactions with high con- mutation analysis. Here we describe a novel mitochondrial cordance to singleplex PCR (R2 = 0.96). copy number assessment approach in a single tube multi- Here we describe a novel single-tube multiplex plex qPCR assay based on molecular beacon probes. Since approach for mtDNA quantitation that provides accurate the mtDNA molecule in each sample may exhibit variance and reproducible results independent of template quality depending on DNA extraction and handling, the major and quantity. The molecular beacon based assay is ame- obstacle in PCR based relative copy number assessment is nable for routine analyses, saving up consumables and the normalization of target sequence. Yet, the best practice time with less effort. is to normalize to a second amplicon that is known to be consistent in all samples. In gene expression studies, this Acknowledgement: This manuscript describes author’s is achieved by normalizing the gene of interest to a reliable efforts to improve current diagnostic laboratory service house-keeping gene. In the case of mtDNA quantitation, quality. Authors are grateful to all physicians and clinical the mtDNA amount is normalized to the diploid genome associates who refer suspected mitochondrial cytopathy amount within the sample. patients for routine testing. Quantitation of mtDNA is of value in the support for the diagnosis of mtDNA cytopathies resulting from genetic Ethical considerations: This study was approved by defects causing mtDNA maintenance. The mtDNA content the local ethics committee of Hacettepe University (GO of each cell is highly variable depending on the tissue 17/519-08). type, oxidative capacity and energy demand. In skeletal Conflict of interest: The authors declare that they have no muscles, mtDNA content is further variable depending on conflict of interest to declare regarding the publication of the muscle fiber composition besides the age and athletic this article. fitness of the individual. 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