A1 Refereed original research article in a scientific journal
Assessing reliability and accuracy of qPCR, dPCR and ddPCR for estimating mitochondrial DNA copy number in songbird blood and sperm cells
Authors: Bagdonaitė, Laima; Leder, Erica H.; Lifjeld, Jan T.; Johnsen, Arild; Mauvisseau, Quentin
Publisher: PeerJ
Publication year: 2025
Journal: PeerJ
Journal name in source: PeerJ
Journal acronym: PeerJ
Article number: e19278
Volume: 13
eISSN: 2167-8359
DOI: https://doi.org/10.7717/peerj.19278
Web address : https://doi.org/10.7717/peerj.19278
Mitochondrial DNA (mtDNA) copy number varies across species, individuals, and cell types. In birds, there are two types of cells with a relatively low number of mitochondria: red blood cells and spermatozoa. Previous studies investigating variation of mitochondrial abundance in animal sperm have generally used quantitative PCR (qPCR), but this method shows potential limitations when quantifying targets at low abundance. To mitigate such issues, we investigated and compared the reliability and accuracy of qPCR, digital PCR (dPCR) and droplet digital PCR (ddPCR) to quantify high and low concentration DNA. We used synthetic DNA targets, to calculate the limit of detection and the limit of quantification and found that with both dPCR and ddPCR, these limits were lower than with qPCR. Then, to compare quantification accuracy and repeatability, we used DNA extracted from blood and sperm cells of Eurasian siskin. We found that qPCR, dPCR and ddPCR all reliably quantified mitochondrial DNA in sperm samples but showed significant differences when analyzing the typically lower levels of mtDNA in blood, with ddPCR consistently showing lower variation among replicates. Our study provides critical insights and recommendations for future studies aiming to quantify target mtDNA and indicates that dPCR and ddPCR are the preferred methods when working with samples with low abundance of mtDNA.
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Funding information in the publication:
Financial support was received from the Research Council of Norway (grant number 301592). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
