Validation of Differentially Expressed Genes by qRT-PCR

Twenty-one genes, including 15 DEGs, were randomly selected, and 6 Pb-regulated genes were used for further validation by qRT-PCR. Total RNA extraction and genomic DNA removal were performed as described above. First-strand cDNA was synthesized from 1 μg of RNA using a HiScript® 1st Strand cDNA Synthesis kit (Vazyme, China). The cDNA products were then diluted tenfold with nuclease-free water for use as a template for qRT-PCR, which was performed using the ChamQ™ SYBR Green Master mix (Vazyme, China) with a CFX96 Real-Time PCR Detection System (Bio-Rad). The specific primer pairs used for qRT-PCR are listed in Additional file 8: Table S5. For qRT-PCR validation, the primer specificity was tested by PCR (Additional file 9: Figure S4). All samples were normalized to the CACS gene [73 (link)]. The DEG expression fold change was calculated based on the threshold cycle (Ct), where ΔCt = Ct_target − Ct_CACS and Δ (ΔCt) = ΔCt_Control − ΔCt_{Indicated condition}.