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Extracting Hormones and DNA

Extracting Hormones and DNA. Blood. Red blood cells Birds: nucleated Mammals: non-nucleated White blood cells Plasma Fluids. Steroid Hormones. Precursor is cholesterol: steroid hormones are fat-soluble, not water-soluble Types of steroid hormones: Androgens (e.g., testosterone)

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Extracting Hormones and DNA

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  1. Extracting Hormones and DNA

  2. Blood • Red blood cells • Birds: nucleated • Mammals: non-nucleated • White blood cells • Plasma • Fluids

  3. Steroid Hormones • Precursor is cholesterol: steroid hormones are fat-soluble, not water-soluble • Types of steroid hormones: • Androgens (e.g., testosterone) • Progestins • Estrogens • Corticoids (e.g., corticosterone)

  4. Extraction of Steroid Hormones from Blood • Separate plasma from RBCs • Diethyl ether extraction method • Diethyl ether separates liquid phase of plasma • Snap-freeze: pour off unfrozen ether with steroid hormones • Dry and reconstitute

  5. Chromatography • To study multiple hormones: you will need to isolate each one from this extract • Run the mixture through layers of celite with varying concentrations of ethyl acetate in isooctane

  6. Extraction from Yolks • Yolk is laid down in layers; maternal steroid concentrations differ in each layer • In nonviable eggs: • Separate yolk from white (usually by freezing) • Homogenize yolk (if measuring total concentration) or sample layers • Extraction protocol similar to blood • Use petroleum ether in addition to diethyl ether: separates out lipids as well

  7. Extraction from Feces • Requires additional steps at the beginning of process (drying, pulverizing) • Additional concerns: metabolizing of steroids

  8. Measuring Extraction Efficiency • In any extraction method, you lose a certain amount of the target material • Add known amount of radioactive hormone before extraction • Measure remaining amount of radioactivity after extraction • Gives you a percentage, and you can correct your estimates of “native” hormone accordingly

  9. Types of Assays • EIA: Enzyme ImmunoAssay • RIA: RadioImmunoAssay • Both work on the principle of competitive binding

  10. Competitive Binding • Sample has “native” hormone • Add to sample either radioactive hormone or alkaline phosphatase bound to hormone • Antibody • Competitive binding: native hormone binds to antibody better than nonnative • Measure remaining amount of nonnative hormone: inversely proportional to native hormone in sample

  11. Setting up an Assay • Randomizing samples

  12. Setting up an Assay

  13. Standard Curve

  14. Standards • Known concentrations of hormone • Used as a sample at beginning, middle, and end of samples

  15. DNA Extraction from Blood • Lysis: break open cells • Proteinase K: breaks up proteins • Phenol/Chloroform: removes proteins and other material • Can also use kits

  16. DNA Extraction from Feces • Shed cells from epithelial lining of intestine • A few difficulties: • Lower-quality DNA • PCR inhibitors in feces, such as bile acids

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