Molecular Sexing of Birds Using Quantitative PCR (qPCR) of Sex-Linked Genes and Logistic Models

Prior to DNA extraction, we homogenized blood samples using PowerBead Pro Tubes (Qiagen Inc.) following the manufacturer’s instructions. DNA was extracted with a Qiacube using the DNeasy Blood and Tissue kit (Qiagen Inc.), after overnight lysis with proteinase K in a shaking incubator at 56°C. DNA concentration and quality were estimated using a spectrophotometer (NanoDrop Technologies) and verified with a Qubit fluorometer if concentration was < 10 ng/µL. All samples were normalized to a concentration of 2 ng/µL. We quantified six Z-linked and three autosomal gene targets for each individual using qPCR. Standard curves ranging from 10,000,000 copies/µL to 1 copy/µL were generated for each gene target using synthetic DNA templates (Integrated DNA Technologies). Amplification reactions were conducted in 20 µL volumes containing 10 µL of 2X SYBR Green PCR MasterMix (Applied Biosystems), 3 µL of ultra-pure water, 0.5 µM of each forward and reverse primer, and 10 ng of template DNA. Thermal cycling was performed on a Bio-Rad CFX96, using these conditions: 50°C for 2 min, 95°C for 3 min, followed by 45 cycles of 95°C for three seconds, 60°C for 30 seconds, and an extension at 72°C for 10 seconds.