Gene expression analysis was performed as previously described.22 (link) To ensure that no cross-effect between challenges was observed, every O-ring of each embryo was infused with the same stimulus. After 12 and 24 hrs incubation at 37°C, 99% (v/v) relative humidity, CAMs were excised and transferred to a microfuge tube. Total RNA was extracted using NZYol reagent (NZYtech, Lisbon, PT), according to the manufacturer's instructions. cDNA synthesis and amplification of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH coding for GAPDH), interleukin-8 (IL8 coding for IL-8), vascular endothelial growth factor A (VEGFA coding for VEGFA) , and vascular endothelial growth factor receptor 1 (FLT1/VEGFR1 coding for VEGFR-1) were performed as previously described, using NZY qPCR green master mix (NZYtech).22 (link)
IL8, VEGFA, and VEGFR1 expression were analyzed using the Ct method (2 −∆∆Ct), with GAPDH as housekeeping gene and samples treated with PBS as control.29 (link) It was considered as significantly altered expression when 2 −∆∆Ct was higher than 2 or lower than 0.5.
IL8, VEGFA, and VEGFR1 expression were analyzed using the Ct method (2 −∆∆Ct), with GAPDH as housekeeping gene and samples treated with PBS as control.29 (link) It was considered as significantly altered expression when 2 −∆∆Ct was higher than 2 or lower than 0.5.
