Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

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Abstract

Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a “dilution bias” when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

Highlights

► Mitochondrial dysfunction is central to many diseases of oxidative stress. ► 95% of the mitochondrial genome is duplicated in the nuclear genome. ► Dilution of untreated genomic DNA leads to dilution bias. ► Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content.

Introduction

Mitochondria are organelles present in most eukaryotic cells in variable numbers ranging from hundreds to thousands of copies per cell and contain their own extra-chromosomal genome, a 16.6 kb circular molecule of double stranded DNA [1]. An individual mitochondrion can contain more than one mitochondrial genome, the number has been estimated to be between 0 and 11 copies with a mean of 2.0 [2]. The amount of mitochondrial DNA (MtDNA) in a cell could provide a major regulatory point in mitochondrial activity, as the transcription of mitochondrial genes is proportionate to their copy numbers [3], [4]. As mitochondria are the major producers of intracellular reactive oxygen species in the cell through free radical generation, a side product of oxidative phosphorylation, it is possible that alteration in MtDNA content could lead to a change in mitochondrial gene transcription and activity and thereby could affect the redox balance of the cell [5].

The role of mitochondrial dysfunction in numerous diseases is well documented with more than 10,000 cited entries in PubMed, NCBI. Studies looking specifically at alterations in MtDNA content in various cell types cover a broad range of human diseases, such as diabetes and its complications [5], [6], [7], [8], [9], [10] obesity [11] cancer [12], [13], [14], [15], [16], [17], [18], [19], HIV complications [20], [21], [22], [23], [24], eye disease [25], [26], nasal polyp [27] and others. In addition, links between altered MtDNA content and development [28], [29], [30], fertility [31], [32], [33], ageing [34], environment [35], [36], and exercise [37], [38], [39] have been shown. Clearly there is a widespread interest in accurately quantifying human MtDNA in a broad spectrum of human diseases. In order to determine if MtDNA content is a potential biomarker of mitochondrial dysfunction, it is important to validate methods for accurate and reproducible measurement of cellular MtDNA content.

A common method for measuring MtDNA content is to quantify a mitochondrial encoded gene relative to a nuclear encoded gene to determine the mitochondrial genome to nuclear genome ratio (Mt/N) using real time qPCR. However in many cases the methodology being used suffers from at least one of the following problems: Firstly, as much of the mitochondrial genome is duplicated in the nuclear genome [40], [41] many MtDNA specific primers co-amplify homologous pseudogenes found in the nuclear genome; secondly, for the quantification of the nuclear genome many studies utilise regions from repetitive and/or highly variable genes such as 18S rRNA which can result in co-amplification errors; thirdly, dilution of genomic template DNA can introduce significant errors in Mt/N values as MtDNA and nuclear DNA do not dilute equally, an effect we have previously described as “dilution bias” and which may be a consequence of the fact that the mitochondrial genome is a circular molecule of 16.5 kb whereas the nuclear genome is composed of linear molecules (chromosomes) of more than 3 million kb in size [5].

In the current paper we describe improvements to the PCR-based method for quantification of MtDNA. MtDNA copy numbers were determined by identifying and amplifying a short unique region of MtDNA not present as a nuclear pseudogene. Nuclear DNA content was determined by amplification of a segment of unique single copy nuclear gene. In addition, we show the differing effects of template dilution on MtDNA and nuclear DNA and describe a protocol for removing dilution bias.

Section snippets

Identification of unique regions of the mitochondrial genome and primer/probe design

The duplication of the mitochondrial genome in the nuclear genome was detected using BLAST (http://www.ncbi.nlm.nih.gov) [42]. Unique regions were identified in the human mitochondrial sequence, retrieved from ENSEMBL [43] using FASTA version 3.5.2.7 [44] as follows: The mitochondrial sequence was split into overlapping fragments of length 150 bp with a 50 bp overlap and each fragment was used as a query sequence in a FASTA search against the entire human genome, one chromosome at a time (both

Duplication of the human mitochondrial genome in the nuclear genome

Using blastn, we compared the mitochondrial genome with the nuclear genome and found that more than 97% of the MtDNA sequence shows homologies with regions in the nuclear genome (Fig. 1A). More than 200 regions of the nuclear genome contained significant matches to the mitochondrial genome, and these matches were scattered throughout the nuclear genome (Fig. 1B).

Identification of unique regions of the mitochondrial and nuclear genome

The entire mitochondrial genome sequence was split into fragments of 150 bp long overlapping by 50 bp and used to identify a unique

Discussion

In the last decade with the wider availability of real time qPCR, there has been a substantial increase in publications reporting changes in MtDNA content in human cells from tissues and circulating cells, and changes in MtDNA have been reported for a wide range of human diseases from cancer to diabetes as well as in development, ageing and exercise [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29],

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