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. 2018 Jul 30;8:11392. doi: 10.1038/s41598-018-29621-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Copy number corrected ACTB provides increase precision and greater dynamic range of than a single copy number genomic locus. (A) Example droplet intensities of A7r5 gDNA amplified with ACTB (BA) using EvaGreen (BA-EG) and EIF2C1-HEX assay in the presence of EvaGreen. (B) BA:EIF2C1 ratio was calculated by linear regression from two independent experiments amplifying varying amounts of gDNA (experiment #1: 3 × 1.0, 2 × 3.0, 2 × 10.0 ng gDNA. Experiment #2: 2 × 2.0, 2 × 4.0 and 2 × 7.0 ng gDNA). Correlation coefficient (r) = 0.997, deviation of slope from zero, p < 0.05. (C) Comparison of BA and EIF2C1 as reference. Multiplex: BA (n = 14), EIF2C1 (n = 24). Singleplex: DL2 (n = 18), BA (n = 5), EIF2C1 (n = 21). (D) Calculation of mtDNA copy number using ACTB and EIF2C1 as reference genes adjusting for ACTB pseudogenes and 2 N ploidy via Multiplex Assay (n = 14 for ACTB, n = 24 for EIFf2C1). (E) Usable range of initial nuclear DNA concentration (copies/µl) in duplex ddPCR reactions for tissues with 300–3000 mtDNA copies/cell using EIF2C1 or BA as nuclear reference.