1. Extracting Hormones and DNA

2. Blood Red blood cells White blood cells Plasma 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