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)– Progestins– Estrogens– Corticoids (e.g., corticosterone)

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

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

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

Extraction from Feces

• Requires additional steps at the beginning of process (drying, pulverizing)

• Additional concerns: metabolizing of steroids

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

Types of Assays

• EIA: Enzyme ImmunoAssay• RIA: RadioImmunoAssay• Both work on the principle of

competitive binding

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

Setting up an Assay

• Randomizing samples

Setting up an Assay

Standard Curve

Standards

• Known concentrations of hormone• Used as a sample at beginning,

middle, and end of samples

DNA Extraction from Blood

• Lysis: break open cells• Proteinase K: breaks up proteins• Phenol/Chloroform: removes

proteins and other material

• Can also use kits

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