RNA extraction and RT-qPCR assay

RNA extraction and RT-qPCR procedures were conducted similarly to that previously described (Noor et al., 2019). Briefly, frozen tissues were retrieved on dry ice and scraped into homogenization tubes or onto a petri dish on dry ice for cutting, ensuring that tissues remained frozen. Total RNA was extracted using the miRNeasy micro kit (Qiagen, catalog # 217084). The petri dish, cutting blade, and forceps were thoroughly cleaned with ethanol, RNAse zap, and DNAse away (Thermo Scientific, MA, United States). The entire tissue sample from the HYP and AMG was processed. A randomly selected half of the HPC within each experimental condition was processed for RNA extraction. Qiazol (200 μl) was added to homogenization tubes containing the frozen tissue, homogenized on ice for 90 s followed by further addition of Qiazol (500 μl), vortexed for 30 s, and incubated at RT for 7-min, which allows dissociation of nucleoprotein complexes, and finally adding 140 μL of chloroform (Sigma-Aldrich; Cat#C2432). The samples were then mixed vigorously for 15 s, incubated for 4 min at RT, mixed vigorously for 10 s, and then centrifuged at 4°C at 12000 × g for 15 min. Approximately 300 μl of the aqueous layer was retrieved from the samples and the final elution of RNA was in 20 μl of RNAse/DNase free sterile water. RNA concentration was assessed using NanoDrop (Thermo Scientific, MA, United States). The remaining sample (~300ul) was aliquoted into homogenization DNase/RNase/Protease-free 1.5 mL disposable pellet mixer microtubes (VWR International; Cat#47747-358), briefly spun down and frozen at − 80 °C until protein assay from the AMG.