Induction of the common deletion in mitochondrial DNA by γ radiation and absolute quantification by real-time PCR

The purpose of this study was to map the common deletion (CD) area in mtDNA and investigate the levels of this deletion in irradiated heart. The assays were developed in male Wistar rats that were irradiated with three different single doses (5, 10 or 15 Gy) delivered directly to the heart and the analyses were performed at various times post-irradiation (3, 15 or 120 days). The CDs area were sequenced and the CD quantified by real-time PCR. Our study demonstrated that the CD levels progressively decreased from the 3rduntil the 15th day after irradiation, and then increased thereafter. Additionally, it was observed that the levels of CD are modulated differently according to the different categories of doses (moderate and high). This study demonstrated an immediate response to ionizing radiation, measured by the presence of mutations in the CD area and a decrease in the CD levels.

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Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

Journal of Food Protection ◽

10.4315/0362-028x-68.6.1217 ◽

2005 ◽

Vol 68 (6) ◽

pp. 1217-1221 ◽

Cited By ~ 27

Author(s):

PAVEL KRCMAR ◽

EVA RENCOVA

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Single Species ◽

Absolute Quantification ◽

Quantitative Detection ◽

Vertebrate Species ◽

Target Species ◽

Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

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Technical report: Laser microdissection of cochlear structures from celloidin embedded human temporal bone tissues and detection of the mitochondrial DNA common deletion using real time PCR

Hearing Research ◽

10.1016/j.heares.2008.07.007 ◽

2008 ◽

Vol 244 (1-2) ◽

pp. 1-6 ◽

Cited By ~ 7

Author(s):

Adam Markaryan ◽

Erik G. Nelson ◽

Maria Tretiakova ◽

Raul Hinojosa

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Temporal Bone ◽

Real Time Pcr ◽

Laser Microdissection ◽

Technical Report ◽

Human Temporal Bone ◽

Common Deletion

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Measurement of the Induction of the 4977‐bp Common Deletion in Human Mitochondrial DNA by Gamma Radiation Using Real‐time PCR

The FASEB Journal ◽

10.1096/fasebj.21.5.a666-b ◽

2007 ◽

Vol 21 (5) ◽

Author(s):

Michael Miller ◽

Jessica Gucwa ◽

Robert O'Donnell ◽

Wendy Pogozelski

Keyword(s):

Mitochondrial Dna ◽

Gamma Radiation ◽

Real Time ◽

Real Time Pcr ◽

Human Mitochondrial Dna ◽

Common Deletion

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A novel method to quantify deleted mitochondrial DNA in a real time PCR

International Congress Series ◽

10.1016/j.ics.2005.09.150 ◽

2006 ◽

Vol 1288 ◽

pp. 756-758 ◽

Cited By ~ 1

Author(s):

T. Schwark ◽

C. Fisch-Kohl ◽

N. von Wurmb-Schwark

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Real Time Pcr ◽

Novel Method

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Real-Time PCR Detection of Ruminant DNA

Journal of Food Protection ◽

10.4315/0362-028x-67.3.550 ◽

2004 ◽

Vol 67 (3) ◽

pp. 550-554 ◽

Cited By ~ 36

Author(s):

LUIS MENDOZA-ROMERO ◽

EDWARD L. C. VERKAAR ◽

PAUL H. SAVELKOUL ◽

ARNOLD CATSBURG ◽

HENK J. M. AARTS ◽

...

Keyword(s):

Mitochondrial Dna ◽

Bovine Spongiform Encephalopathy ◽

Real Time ◽

Real Time Pcr ◽

Spongiform Encephalopathy ◽

Meat And Bone Meal ◽

Bone Meal ◽

Dna Targeting ◽

Species Origin ◽

Semiquantitative Method

To control the spread of bovine spongiform encephalopathy, several DNA methods have been described for the detection of the species origin of meat and bone meal. Most of these methods are based on the amplification of a mitochondrial DNA segment. We have developed a semiquantitative method based on real-time PCR for detection of ruminant DNA, targeting an 88-bp segment of the ruminant short interspersed nuclear element Bov-A2. This method is specific for ruminants and is able to detect as little as 10 fg of bovine DNA. Autoclaving decreased the amount of detectable DNA, but positive signals were observed in feeding stuff containing 10% bovine material if this had not been rendered in accordance with the regulations, i.e., heated at 134°C for 3 instead of 20 min.

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Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

Applied and Environmental Microbiology ◽

10.1128/aem.00035-12 ◽

2012 ◽

Vol 78 (10) ◽

pp. 3630-3637 ◽

Cited By ~ 22

Author(s):

Karol Krak ◽

Martina Janoušková ◽

Petra Caklová ◽

Miroslav Vosátka ◽

Helena Štorchová

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Real Time Pcr ◽

Glomus Intraradices ◽

Large Subunit ◽

Arbuscular Mycorrhizal ◽

Am Fungi ◽

Content Type ◽

Copy Numbers ◽

Pcr Assays

ABSTRACTReal-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates ofGlomus intraradicessensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

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Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR

Nucleic Acids Research ◽

10.1093/nar/gnf067 ◽

2002 ◽

Vol 30 (14) ◽

pp. 68e-68 ◽

Cited By ~ 211

Author(s):

L. He

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Real Time Pcr ◽

Dna Deletions ◽

Detection And Quantification

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Quantification of mRNA in Whole Blood by Assessing Recovery of RNA and Efficiency of cDNA Synthesis

Clinical Chemistry ◽

10.1373/clinchem.2005.048983 ◽

2006 ◽

Vol 52 (4) ◽

pp. 634-642 ◽

Cited By ~ 24

Author(s):

Masato Mitsuhashi ◽

Shigeru Tomozawa ◽

Katsuya Endo ◽

Atsushi Shinagawa

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Whole Blood ◽

Ex Vivo ◽

Statistical Significance ◽

Absolute Quantification ◽

External Control ◽

Cdna Synthesis ◽

Optimized Conditions

Abstract Background: Current gene expression analysis relies on the assumption that the isolated RNA represents all species of mRNA in proportions equal to those in the original materials. No system is available for absolute quantification of mRNA. Methods: We applied whole blood to 96-well filterplates to trap leukocytes. Lysis buffer containing cocktails of specific reverse primers and known concentrations of synthetic external control RNA (RNA34) was added to filterplates, and cell lysates were transferred to oligo(dT)-immobilized microplates for hybridization. We then synthesized the cDNA in the oligo(dT)-immobilized microplates from these primer sites and used the cDNA for real-time PCR. RNA34 acted as a universal control, and gene amplification results were converted to quantities of mRNA per microliter of whole blood after the recovery of RNA34 in each sample was determined. Results: Under fully optimized conditions, both added RNA34 and native mRNA species exhibited ∼10% recovery from whole blood to real-time PCR. When whole blood was stimulated ex vivo, changes in gene expression as low as 30%–40% were detected with statistical significance, and the experimental CVs were low (10%–20%). Conclusion: This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression–based molecular diagnostics.

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