Fig. 6

Confirmation of RNA sequencing gene expression results. (A) Confirmation by RT-qPCR of hypothalamic gene expression as determined by RNA sequencing. Six differentially expressed genes (DEGs), with expression patterns that showed inverse regulation in low egg producing hens (LEPH) and high egg producing hens (HEPH) outside and during the preovulatory surge (PS) [alpha-actin-2 (ACTA2) and transthyretin (TTR)], up-regulation in HEPH both outside and during the PS [biogenesis of lysosomal organelles complex-1, subunit 4 (BLOC1S4) and radical s-adenosyl methionine domain containing 2 (RSAD2)], and up-regulation in LEPH both outside and during the PS [mannosidase endo-alpha (MANEA) and neuropeptide VF precursor (NPVF)], as well as two genes that were not differentially expression in LEPH and HEPH either outside or during the PS [integral membrane protein 2A (ITG2A) and proteasome subunit alpha 2 (PSMA2)] were confirmed through RT-qPCR. Normalized data are presented relative to LEPH expression outside of the PS for each gene. Significant expression differences between LEPH and HEPH for a given condition are denoted with an asterisk (*). (B) Confirmation by RT-qPCR of pituitary gene expression as determined by RNA sequencing. Six DEGs, with expression patterns that showed inverse regulation in LEPH and HEPH outside and during the PS [crystallin beta B1 (CRYBB1) and extended synaptotagmin 3 (ESYT3)], up-regulation in HEPH both outside and during the PS [lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) and fatty acid binding protein 6 (FABP6)], and up-regulation in LEPH both outside and during the PS [pappalysin 2 (PAPPA2) and zinc finger protein, FOG family member 1 (ZFPM1)], as well as two genes that were not differentially expression in LEPH and HEPH either outside or during the PS [insulin like growth factor 1 (IGF1) and transforming growth factor beta receptor 1 (TGFBR1)] were confirmed through RT-qPCR. Data are presented relative to LEPH expression outside of the PS for each gene. Significant differences between LEPH and HEPH for a given condition are denoted with an asterisk (*)