Table 1.
Primer sequences of the selected candidate reference genes and their full names and function.
Fig 1.
Raw quantification cycle (Cq) values.
Cq values for each candidate reference gene in the gastrocnemius, diaphragm and heart. Each datapoint represents the mean Cq value of an individual sample for each gene. Data is separated by strain: Light blue: BL10-wt; Dark blue: BL10-mdx; Light red: D2-wt; Dark red: D2-mdx.
Fig 2.
Rankings of the pairwise stability value M of the nine candidate reference genes, assessed by the geNorm method, for (A) the whole dataset, (B) healthy and dystrophic subsets, and (C) tissue-specific subsets. Genes are ranked from low stability (high M value) to high stability (low M value), where M values < 0.5 indicate highly stable genes. The highest scoring genes (the ‘best pair’) are considered to be equal in M value.
Fig 3.
Rankings of the coefficient of correlation (r) of the nine candidate reference genes, assessed by the BestKeeper method, for (A) the whole dataset, (B) healthy and dystrophic subsets, and (C) tissue-specific subsets. Genes are ranked from low stability (low r) to high stability (high r).
Fig 4.
Rankings of the average deltaCt standard deviation of the nine candidate reference genes, assessed by the deltaCt method, for (A) the whole dataset, (B) healthy and dystrophic subsets, and (C) tissue-specific subsets. Genes are ranked from low stability (high deltaCt score) to high stability (low deltaCt score).
Fig 5.
NormFinder rankings (ungrouped).
Ungrouped rankings of the stability value of the nine candidate reference genes, assessed by the NormFinder method, for (A) the whole dataset, (B) healthy and dystrophic subsets, and (C) tissue-specific subsets. Genes are ranked from low stability (high stability value) to high stability (low stability value).
Fig 6.
NormFinder rankings (grouped).
Grouped rankings of the stability value of the nine candidate reference genes, assessed by the NormFinder method. The whole dataset was grouped by (A) disease, (B) tissue, (C) strain, or (D) age. For the gastrocnemius, diaphragm and heart samples, genes were grouped by (E) disease, strain, or age. Genes are ranked from low to high stability (high to low values). For each grouping, the best pair is indicated on the plot.
Fig 7.
Aggregate rankings of the four methods.
Rankings of the geometric mean scores of the nine candidate reference genes from all four analysis methods, for (A) the whole dataset, (B) healthy and dystrophic subsets, and (C) tissue-specific subsets. Genes are ranked from low stability (high geometric mean rank) to high stability (low geometric mean rank).
Table 2.
Overview of the candidate reference genes and their ranking for each analysis method.
Fig 8.
Normalization of Actb and Rpl13a.
(A) Mean raw RQ values for Actb in gastrocnemius, diaphragm and heart. (B) Normalization to the geometric mean of Htatsf1, Pak1ip1 and Zfp91 in gastrocnemius, diaphragm and heart shows reduced variation. (C) Mean raw RQ values for Rpl13a in gastrocnemius, diaphragm and heart. (D) Normalization to the geometric mean of Htatsf1, Pak1ip1 and Zfp91 in gastrocnemius, diaphragm and heart shows reduced variation. Data is shown as log10 of RQ values. CoV values represent the average of the individual CoVs per time-point, per tissue. Data is separated by strain: Light blue: BL10-wt; Dark blue: BL10-mdx; Light red: D2-wt; Dark red: D2-mdx.