*3.9. Validation of Reference Genes*

To verify the reliability of the selected reference genes, the transcripts of two genes were quantified using either different combinations of the two most stable genes, using a single reference gene or using the relatively unstable reference gene *TUA* under six different experimental conditions. The test genes were *NAC* and *ERF*, both of which show relatively high abundance levels, with fragments per kilobase of transcript per million fragments mapped (FPKM) values of approximately 22–223. The relative expression patterns and levels of the *LmNAC83* gene showed similar trends when the most stable genes were used alone or in combination as reference genes for standardization (Figure 7A–F). In contrast, the relative expression levels of *LmNAC83* showed significant fluctuations when the relatively unstable gene *TUA* was used for relative quantification. For example, under heat treatment for 24 h, the expression levels of *LmNAC83* in leaves was the highest at 30 ◦C when using the most stable genes as the reference genes. However, the relative expression of *LmNAC83* was low when using the unstable gene (*TUA*) as the reference gene (Figure 7F). In addition, the expression levels and trends of the *LmERF60* gene (Figure 8) were very similar to those found in the analysis of *LmNAC83*. It is evident that the use of unstable internal reference genes for gene expression analysis in *L. megaphylla* can lead to unreliable results. This test illustrates the importance of screening stable internal reference genes under different experimental conditions.

**Figure 7.** Relative expression of *LmNAC83* in six experimental sample sets. The results were normalized using the stable reference genes (alone or in combination) or an unstable gene in six sample sets, including (**A**) different seedling tissues; (**B**) different adult tree tissues; (**C**) different leaf developmental stages; (**D**) cold treatment for 7 days; (**E**) cold treatment for 24 h; and (**F**) heat treatment for 24 h. The bars indicate the standard deviation (±SD) evaluated from three biological replicates.

**Figure 8.** Relative expression of *LmERF60* in six experimental sample sets. The results were normalized using the stable reference genes (alone or in combination) or an unstable gene in six sample sets, including (**A**) different seedling tissues; (**B**) different adult tree tissues; (**C**) different leaf developmental stages; (**D**) cold treatment for 7 days; (**E**) cold treatment for 24 h; and (**F**) heat treatment for 24 h. The bars indicate the standard deviation (±SD) evaluated from three biological replicates.
