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2.2. Tissue samples
2.2.1. Study one
Following euthanasia of mature WL hens, tissue samples were collected from brain, heart, liver, kidney, small intestine, gizzard, ovary, oviduct and testis of 1–2 year-old males (n = 3) and females (n = 3).
2.2.2. Study two
Female chicks were identified by PCR analysis using W chromosome-specific primer sets [9]. Treatment with DES and recovery of TG-101209 oviduct (n = 5) were conducted as reported previously [10] and [11].
2.2.3. Study three
A total 136 chickens (88 chickens aged over 36 months and 48 chickens aged over 24 months), which had completely stopped laying eggs were euthanized for biopsy and cancerous (n = 10) ovaries were collected. As a control, normal (n = 5) ovaries were also collected from egg-laying hens. We examined the tumor stage in 10 chickens with cancerous ovaries using characteristic features of chicken ovarian cancer [12] and [13].
2.3. RNA isolation
Total cellular RNA was isolated from frozen tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s recommendations.