Fig 1.
Specificity of RT-qPCR amplification.
Melting curves (dissociation curves) of the 13 reference genes amplicons after the RT-qPCR reactions, all showing one peak. Melting, amplification and standard curves for mitochondrially encoded cytochrome c oxidase II (MT-CO2) target gene are also showed.
Table 1.
Reference genes, MT-CO2 target gene, specific RT-qPCR primers and different parameters derived from RT-qPCR analysis.
Table 2.
Descriptive statistics and expression level of reference genes obtained by BestKeeper (n = 52).
Table 3.
Normality test (Shapiro-Wilk).
Table 4.
Results of ranking of thirteen reference genes obtained using five different algorithms and overall rank of candidate reference genes for chicken muscle.
Three overall best references genes are indicated in bold.
Fig 2.
Reference genes stability for all treatments.
NormFinder analysis of reference genes in chicken Pectoralis major muscle showing stability numbers.
Fig 3.
Determination of the Optimal Number of Control Genes for Normalization.
This graph show the geNorm Excel pairwise variation (V) analysis. These values were used to determine optimal number of reference genes based on V-value for normalization in RT-qPCR reaction. Each bar represents change in normalization accuracy when stepwise adding more reference gene.
Fig 4.
Spearman correlation matrix between five different stability algorithms.
A correlation matrix was built between BestKeeper, NormFinder, geNorm Excel, geNorm SAS, and ΔCt method. P-value are described are in parenthesis.
Table 5.
Pearson correlation matrix visualizing reference genes ranked by five different stability tests (BestKeeper, NormFinder, geNorm Excel, geNorm SAS, and ΔCt method.
p-value are described are in parenthesis. Significative values are indicated in bold.
Fig 5.
Relative expression of this MT-CO2 target gene when normalized with poor ranked reference genes and the best reference genes using Pfaffl method.
This Figure shows the effect of incorrect selection of reference gene in the gene expression of target gene. Two best ranked reference genes (HMBS and HPRT1) and the two poor ranked reference genes (TFRC and B2M). The results also include standard errors (SE_R) and two-tailed P values by Student's t test. A single asterisk indicates P-value < 0.05.
Table 6.
Comparative primer efficiency as calculated by geNorm Excel and geNorm SAS.