Genomic DNA was extracted from TNFα-treated hASM cells and untreated control hASM cells using QIAamp DNA Mini Kit (Cat. No. 51304, Qiagen, Hilden, Germany) as per manufacturer’s protocol and quantified using Nanodrop spectrophotometer (Thermo Fisher Scientific, Rockville, IL, USA). The relative copy number of human mtDNA was quantified using Human Mitochondrial DNA Monitoring Primer Set (Cat. no. 7246, Takara Bio USA, Mountain View, CA, USA). Briefly, genomic DNA from TNFα-treated hASM cells and untreated control hASM cells was subjected to quantitative real-time PCR (qPCR) using SYBR green master mix (Cat. No. 04707516001, LightCycler® 480 SYBR Green I Master, Roche Scientific, Thermo Fisher Scientific, Rockville, IL) as per the manufacturer’s instructions. The primer set contains two nuclear gene-specific primers, solute carrier organic anion transporter family member 2B1 (SLCO2B1) and serpin family A member 1 (SERPINA1)]; and two mtDNA specific primers, NADH dehydrogenase subunit 1 (ND1) and NADH: ubiquinone oxidoreductase core subunit 5 (ND5)] (Cat. no. 7246, Takara Bio USA, Mountain View, CA, USA). The relative quantification of mtDNA copy number was represented as the difference in cycle threshold (Ct) values for mtDNA and nuclear DNA.
Human mitochondrial dna monitoring primer set
Manufactured by Takara Bio
Sourced in United States, Japan
The Human Mitochondrial DNA Monitoring Primer Set is a laboratory equipment product designed for the detection and quantification of human mitochondrial DNA. It provides the necessary components to perform targeted PCR amplification and analysis of specific mitochondrial DNA regions.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Mitochondrial dna isolation kit, by Abcam (1 mentions)
Agencourt ampure xp system, by Beckman Coulter (1 mentions)
Takara minibest universal genomic dna extraction kit, by Takara Bio (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Nucleospin tissue, by Macherey-Nagel (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
Thermal cycler dice real time system 3, by Takara Bio (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
8 protocols using human mitochondrial dna monitoring primer set
1
Quantifying Mitochondrial DNA Copy Number in hASM Cells
2
Quantifying Mitochondrial DNA Content
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The mitochondrial DNA (mtDNA) copy number of HDFs was measured by real-time polymerase chain reaction with Human Mitochondrial DNA Monitoring Primer Set (Takara, Seoul, Korea). Total mtDNA from HDFs (young, senescent, and Rg3(S)-pretreated senescent cells) was extracted with Mitochondrial DNA Isolation Kit (BioVision, Milpitas, CA), as described previously [22] (link). Then, mtDNA was enriched with Agencourt AMPure XP system (Beckman Coulter, Brea, CA) and subjected to polymerase chain reaction quantification using MightyAmp™ for Real Time (TB GreenTM Plus, Takara, Seoul, Korea). The relative quantification of either senescent HDFs or Rg3(S)-pretreated senescent HDFs divided by young HDFs was calculated using the formula relative quantification = 2(−ΔΔCt) and finally expressed as copy numbers of mtDNA.
3
HUVEC Mitochondrial DNA Quantification
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Total DNA from HUVECs was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa) and quantified using NanoDrop 2000 (Thermo Fisher Scientific). Mitochondrial DNA level was detected using the Human Mitochondrial DNA Monitoring Primer Set (TaKaRa) with q-PCR. The procedures for DNA extraction and mtDNA quantification were conducted according to the manufacturer's manual.
4
Mitochondrial DNA Copy Number Quantification
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After drug treatment, cells were washed with 1 × PBS (-) and then the genome was purified using DNeasy Blood and Tissue Kits (Qiagen). The copy number of mtDNA was measured using a Human Mitochondrial DNA Monitoring Primer set (Takara Bio) according to the manufacturer's instructions.
5
Quantifying Mitochondrial DNA Content
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Total DNA was extracted using NucleoSpin Tissue (MACHEREY-NAGEL, Düren, Germany) from the mouse hearts or the cells cultured in six-well plate following manufacturer protocol. The DNA concentration was measured using NanoDrop 2000 (Thermo Fisher Scientific) and 5 ng/μl of DNA was subjected to Real-Time PCR analysis (Bio-Rad) using Mouse Mitochondrial DNA Copy Number Assay kit (Detroit R&D, Detroit, MI) for the samples from the mouse heart or Human Mitochondrial DNA Monitoring Primer Set (TaKaRa Bio) for the cells following manufacturer protocol.
6
Measuring Mitochondrial DNA Abundance
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MtDNA abundance was measured with the ΔΔCt method as previously described97 (link). Briefly, ~15 mg of frozen liver or muscle tissues were used to extract total cellular DNA using NucleoSpin Tissue kit (Macherey-Nagel, 740952). For qPCR, 20 ng of DNA was used. qPCR was carried out as described above. For mtDNA amplification, primers against the 16S rRNA were used (Supplementary Table 6 ). For nuclear DNA amplification primers against the β2M gene were used (Supplementary Table 6 ). For mtDNA copy number estimation in human myoblasts the Human Mitochondrial DNA Monitoring Primer Set (Takara, 7246) was used.
7
Mitochondrial DNA Copy Number Quantification
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Tissue and DNA were extracted as per the mtDNA NGS methodology below, using the same tissue cut sizes and quantities. The Human Mitochondrial DNA Monitoring Primer Set (Takara Bio, Cat # 7246) was used to determine mtDNA copy number. Analysis was performed as per the manufacturers' instructions.
8
Quantifying Mitochondrial DNA in MRC-5 Cells
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Total DNA was isolated from MRC-5 cells using DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany). To quantify copy number of mtDNA, we performed qPCR analyses using Human Mitochondrial DNA Monitoring Primer set (TAKARA BIO, Shiga, Japan) and Thermal Cycler Dice Real Time System III (TAKARA BIO).
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