Computational protocol: Development of a qPCR Method to Measure Mitochondrial and Genomic DNA Damage with Application to Chemotherapy-Induced DNA Damage and Cryopreserved Cells

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Protocol publication

[…] Four oligonucleotide primer sets were designed using Primer3 [] for a long nuclear gene target of 3129 bp in the E2F transcription factor 1 (E2F1) gene (accession no: AF516106.1; 3427–6565 bp) and a long mitochondrial target 3723 bp (accession no: NC_012920; 11492–15214 bp). For each long amplicon target, a matched reverse primer for an internal short amplicon of between 50 and 150 bp was also designed (). All primer sequences were tested using the primer blast web tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to ensure primer specificity.qPCR analysis was performed using a Corbett Rotor-Gene 6000 instrument (Corbett Research, Mortlake, Australia). All reaction components were optimized through extensive trialing. Final conditions were: 1× buffer B1, 2.5 mM MgCl2, 1 U Hot FIREPol® DNA Polymerase (Solis Biodyne, Tartu, Estonia), 200 µM dNTPs (Genscript, Piscataway, NJ, USA) and 2 µM SYTO82 fluorophore (Life Technologies, USA) in a 20 µL reaction volume, which also contained primer at a concentration of 500 nM and 25–50 ng of DNA template (diluted in MQ sterile water). Each reaction was carried out in an Axygen® optically-clear thin-walled PCR tube (Corning, Tewksbury, MA, USA) in a 36-well rotor. The optimized cycling conditions () included a Hotstart step (10 cycles with a 0.5 °C decrease per cycle) and an extension of 240 s to ensure amplification of the long product.PCR amplification efficiencies for the target amplicons were calculated by comparative quantitation using the Corbett Rotor-Gene 6000 Application Software, version 1.7 (Qiagen,Valencia, CA USA). Average efficiencies for both large and small products across each experimental series were used for these calculations. Detected lesion rate per 10 kb was determined using the following equation modified from Lehle et al. []: Lesions per 10 kb = [(ELCpl(Sample) × ES−Cps(Sample)/ELCpl(Control) × ES−Cps(Control))1/a − 1] × 10,000 where EL and ES are the average amplification efficiencies of the large and short product, Cp values are the crossover (or threshold) values determined by the Rotor-Gene software (Cpl: long; Cps: short), and a is the number of base pairs of the long fragment. All qPCR reactions for each sample were carried out in duplicate, and the mean Cp values were used in the calculation of lesions in either genomic or mitochondrial DNA. An Excel spreadsheet for the above calculation is included in the supplementary materials (). […]

Pipeline specifications

Software tools Primer3, Primer-BLAST
Application qPCR
Diseases Neoplasms
Chemicals Bleomycin, Cisplatin