Quantification of Neurospora crassa mitochondrial DNA using quantitative real‐time PCR

2020 ◽  
Vol 71 (2) ◽  
pp. 171-178
Author(s):  
J. Monteiro ◽  
A. Videira ◽  
F. Pereira
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr

scholarly journals Lamivudine/telbivudine-associated neuromyopathy: neurogenic damage, mitochondrial dysfunction and mitochondrial DNA depletion

2014 ◽  
Vol 67 (11) ◽  
pp. 999-1005 ◽  
Author(s):  
Hongliang Xu ◽  
Zhaoxia Wang ◽  
Lemin Zheng ◽  
Wei Zhang ◽  
He Lv ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Dysfunction ◽  
Real Time ◽  
Real Time Pcr ◽  
Axonal Neuropathy ◽  
Nerve Biopsy ◽  
Quantitative Real Time Pcr ◽  
Electronic Microscopy ◽  
Nucleotide Analogues ◽  
Mitochondrial Dna Depletion

AimsMyopathy or neuropathy has been associated with lamivudine/telbivudine therapy in hepatitis B patients. We aim to describe the pathological changes of lamivudine/telbivudine-associated neuromyopathy.MethodsWe retrospectively recruited six patients who were diagnosed with nucleotide analogues-associated myopathy or neuropathy. Muscle and nerve biopsy were performed, and the specimens were prepared for the light microscopy and electron microscopy. Genomic DNA was extracted from frozen muscle specimens, and the mitochondrial DNA (mtDNA) content was quantified by real-time PCR.ResultsRecovery of the myopathy can be achieved after the discontinuation or changing the drugs to entecavir. Muscle and nerve biopsy revealed similar changes under either the light or electronic microscopy in all the subjects. Quantitative real-time PCR revealed decrease of mtDNA content in the affected muscle.ConclusionsMtDNA depletion results in mitochondrial dysfunction in the lamivudine/telbivudine-associated neuromyopathy. Myopathy was characterised by mitochondrial dysfunction accompanied with neurogenic damage due to axonal neuropathy. Ultrastructure changes of mitochondria included vacuolisation, simplification of the cristae and homogenised matrix.

scholarly journals Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA

2017 ◽  
Vol 10 (1) ◽  
Author(s):  
Barbara A. Qurollo ◽  
Nikole R. Archer ◽  
Megan E. Schreeg ◽  
Henry S. Marr ◽  
Adam J. Birkenheuer ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Molecular Detection ◽  
Diagnostic Assay ◽  
Quantitative Real Time Pcr

Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality

2018 ◽  
Vol 14 (1) ◽  
pp. 70-75 ◽  
Author(s):  
Corey Goodwin ◽  
Denice Higgins ◽  
Shanan S. Tobe ◽  
Jeremy Austin ◽  
Andrew Wotherspoon ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Human Mitochondrial Dna

scholarly journals Heteroplasmy Detection of Mitochondrial DNA A3243G Mutation Using Quantitative Real-Time PCR Assay Based on TaqMan-MGB Probes

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Enguang Rong ◽  
Hanbo Wang ◽  
Shujing Hao ◽  
Yuhong Fu ◽  
Yanyan Ma ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Testing ◽  
Hair Follicles ◽  
Clinical Test ◽  
Nucleotide Position ◽  
Quantitative Real Time Pcr ◽  
A3243g Mutation ◽  
Accurate Quantification

A point mutation of mitochondrial DNA (mtDNA) at nucleotide position 3243 A to G (mt.3243A>G) is involved in many common diseases, including maternally inherited diabetes and deafness (MIDD) and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS). However, the mutant level of mt.3243A>G varies both among individuals and in different organs, tissues, and even cells of single individuals. For detection of this mutation, current methods have limited universality and sensitivity and may be not adequate for a routine clinical test. Here, we develop and evaluate a rapid TaqMan-MGB quantitative real-time PCR (qPCR) method for detecting and quantifying the heteroplasmy level of mt.3243A>G in single-tube analysis. With our method, the sensitivity of detection was as low as 0.1%, but the accuracy of quantification was reliable, down to 4%. All positives could be correctly identified, and the heteroplasmy levels determined by qPCR correlated well with the results from restriction fragment length polymorphism (RFLP) and pyrosequencing assays (r = 0.921~0.973 and 0.972~0.984). In addition, we demonstrated that the urinary sediments, leukocytes, or hair follicles might be ideal templates to detect and quantify the heteroplasmy of mt.3243A>G mutation; however, they should be optimized or retreated for further accurate quantification. Our study should allow rapid and high throughput diagnostic testing and can potentially be used to clarify the association between clinical phenotype and pathogenic mitochondrial mutations derived from various tissues.

scholarly journals Quantitative Real Time PCR (qRT‐PCR) Evaluation of Pig Mitochondrial DNA Damage

2017 ◽  
Vol 31 (S1) ◽  
Author(s):  
Johnny L Barr ◽  
Theo W Thomas ◽  
Ruby S Gibson ◽  
Michael A Dubick ◽  
Phillip D Bowman
Keyword(s):  
Dna Damage ◽  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr ◽  
Mitochondrial Dna Damage

scholarly journals KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yi Wang ◽  
Hongjuan Liao ◽  
Yueheng Wang ◽  
Jinlin Zhou ◽  
Feng Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Mtor Signaling ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

2008 ◽  
Vol 375 (1) ◽  
pp. 150-152 ◽  
Author(s):  
Cheng Xin Yi ◽  
Jun Zhang ◽  
Ka Man Chan ◽  
Xiao Kun Liu ◽  
Yan Hong
Keyword(s):  
Gossypium Hirsutum ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Transgene Copy Number
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