Experimental Analysis

In-vivo - in living organism

Ex-viv - removed from living organism

In-vitro - in lab (in glass)

In situ - in natural location

In silico - in computer

• Deliberate testing directed by previous knowledge
• Two parallel experiments - test (predicted outcome) and control (identical - factor omitted)
• Compare
• Null and test hypothesis 
• Control - tests technique/apparatus/reagents
  • Positive control - expected answer - test for false negatives
  • Negative control - no result - test for false positives
  • Placebo effect - medical - belief in treatment = improvement even when absent
    • Double blind trial - neither doctor nor patient knows = no bias
  • Nocebo effect - drug side effects on placebo = prevents drugs working

Must seperate samples and preserve biological activity/life

Homogenisation

• Homogenate - mixed cell-free suspension
• Mechanical disruption
  • Blender
  • Mortar and pestle
  • Extrusion (syringe needle) - bacteria
  • Homogeniser - crush things to gap of <500um
• Preservation in external aq environment e.g. pH, osmolarity
  • Replicate biological fluids e.g. blood
  • Substitute bioloical fluids e.g. Ringers solution (body conc ions)
  • Prevent drying/denaturation/osmotic shock
  • Keep chilled

• Pellet suspended cells etc - pushed to bottom
• Diagnose leukaemia - num. RBC in pellet
• Sedimentation coefficient - how quickly a molecule pellets = isolate different cell parts (speed)
• Precipitation - difference in solubility add:
  • Salt - compete with other molecules for water hydrogen bonding = precipitation
  • Organic solvents
    • Acetone - precipitates proteins
    • Ethanol - precipitates DNA
  • Insoluble molecules precipitate easily = seperated
• Molecule of interest could be in pellet or supernatant
• Basic centrifugation - heaviest particles settle fastest
• Sucrose density centrifugation - more conc sucrose at bottom, not equilibrium gradient but more effective
• Isopycninc centrifugation (CsCl density gradient) - reacts with water and creates density gradient. Substances pellet = density of CsCl solution
• Ultracentifugatiion - high speed
• Microcentrifugation - small sample
• Reverse centrifugation - mixes things back together again

• Glass tube w/ chemically inert gel beads
• Seperate on size/solubility/charge/biological properties
• Differential elution - passes through at different speeds
• Bead types:
  • Magnetic
    • PolyT bases - bonds with mRNA polyA tail
    • Magnets drag beads to side = collection
    • Purifies mRNA
  • Glass
    • DNA sticks in high salt
    • Centrifuge to remove supernatant
    • Distilled water rinse to elute
• Affinty chromotography - mixture of enzymes and beads coated with enzyme inhibitor

• Generate idea of quantities or presence of mol using specific technique e.g. blood test
• Medical test - compared to reference range

Spectroscopy

• Shine radiation through sample
• Absorbance/emission detected
• Types
  • Concentration/cell density - increased scatter/absorption = decreased reading (can detect flouresence)
  • Molecular identification
  • Molecular changes

Colorimetry

• Proteins - biuret/folin/coomassie blue
• Amino acids - nihydrin
• DNA - diphenylamine
• RNA - bial

• Dose-response curve - response to drug
• Determine concentration required for desired effect