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. 2020 Sep 14;10:15057. doi: 10.1038/s41598-020-71895-x

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Reference gene expression stability analysis in hypothalamus tissue at seven days following surgery. (A) Distribution of threshold cycle (Ct) values for each group (Fx = fracture; TBI = traumatic brain injury; TBI + Fx = combined injury) of all candidate reference genes tested. (B) Pairwise variation (V_n/n+1) analysis by GeNorm, to determine the optimal number of reference genes for normalisation. (C) Stability analysis by NormFinder (stability value: sv), GeNorm (M value) and BestKeeper (coefficient of correlation: r, standard deviation: SD), with a sum displayed in the final rank (green = most stable, red = least stable). (D) Overall expression stability confirmation of RefFinder, which combines NormFinder, GeNorm, BestKeeper and the comparative delta-Ct method.