Quantitative analysis of total mitochondrial DNA: Competitive polymerase chain reaction versus real‐time polymerase chain reaction

Abstract

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.

Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents

“…Five nanograms of total DNA was used as a template in 50 µl PCR reaction using a TaqMan gene expression assay against NADH dehydrogenase 2 (mitochondrial genome [ND2]) and β-actin (nuclear genome). mtDNA copy number was presented as a ratio of ND2 to β-actin as previously described (35).…”

Mitochondrial Gene Expression Changes in Normal and Mitochondrial Mutant Cells after Exposure to Ionizing Radiation

“…Primers and TaqManH probes specific for mitochondrial DNA were obtained from Applied Biosystems (Foster City, CA). Primers and probes specific for the nuclear gene RNase P were used as an internal control and the ratio of mitochondrial DNA to nuclear DNA was used to determine mitochondrial DNA copy number (24). The human RNase P gene exists in a single-copy and encodes the RNA moiety for the RNase P enzyme.…”

Preferential induction of cytochrome P450 1A1 over cytochrome P450 1B1 in human breast epithelial cells following exposure to quercetin

“…After reverse transcription, RNase H (2 units/µl) was added to all samples to ensure degradation of the remaining RNA. Real time PCR was performed in duplicate 25 µl reactions by using the Applied Biosystems (Foster City, CA) 7500 Real Time PCR System as described previously [57]. Data were analyzed using Applied Biosystems 7500 SDS version 1.7 software.…”

“…The expression of Cyp1A1 and Cyp1B1 relative to cyclophilin was determined by dividing the number of cDNA molecules for the gene of interest by the number of cyclophilin cDNA molecules. Standards were created for each gene and a standard curve was run on each plate to allow for accurate quantification of cDNA, as reported previously [56,57].…”