Radiobio 2006

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Radiobio 2006

  1. 1. 2005 Cancer Biology Course:Methods in Molecular Biology Gary D. Kao, M.D., Ph.D. Department of Radiation Oncology
  2. 2. “The progression of scientific enquiry”DNA - “the blue print for mRNA”RNA - “regulated or dysregulated expression”Protein - too much, too little, or mutant proteinsCancer Cells – proliferating, invasive, metastaticAnimal models - xenografts vs. endogenous tumorsPatients - retrospective vs prospective studies
  3. 3. “Standard Molecular Assays”DNA - Southern Analysis (blotting) Polymerase Chain Reaction (PCR) Restriction/ linkage (allelic) analysesRNA - Northern blotting RT (reverse transcriptase)-PCR Real-time PCR Microarray “Gene array”Protein – ELISA Western blotting Immunoprecipitation HPLC Immuno-fluorescence -histochemistryCells – Green fluorescent protein (GFP)-tagging Immunofluorescence Immunohistochemistry
  4. 4. Southern (DNA) and Northern (RNA) Analysis
  5. 5. The Power of PCR:“The Case of theHarmful Hamburger”
  6. 6. Polymerase Chain Reaction (PCRT) & RT-PCR: DNA & RNA
  7. 7. Polymerase Chain Reaction (PCRT) & RT-PCR: DNA & RNA Agarose gel, EtBr -stained
  8. 8. • QUANTITATION OF mRNA – Northern Blotting very cumbersome – Ribonuclease protection assay cumbersome – In situ Hybridization localization – PCR • most sensitive • can discriminate closely related mRNAs • technically simple • but difficult to get truly quantitative results using conventional PCR
  9. 9. Real-Time PCRReal-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production ateach PCR cycle (in real time) as opposed to the endpoint detection
  10. 10. Using the PCR Equation XnXn = X0(1 + E)nXn = PCR product after cycle nX0 = initial copy numberE = amplification efficiencyn = cycle number X0 cycle number
  11. 11. Assessing DNA & RNA Southern/ Microarray PCR Real-time PCR Northern blotting $2000 / $3000 / $30,000 / Core Facility Gel box Machine MachineCOST minimal $20 per expt. $200 per expt. $2000 per expt. Radioactive!CONVENIENCE Takes days easy! Demanding easy! to weeks need PCR/ Semi-CONCERNS Radioactive! Most quantitative Northerns quantitative to confirm!
  12. 12. Enzyme-Linked Immunosorbent Assay (ELISA)
  13. 13. Western blotting: Protein Transfer to a Membrane SDS-PAGE Gel Membrane is probed with Antibody localization – antibody specific for protein of therefore Protein - detected by interest chemilluminescence
  14. 14. Western blotting: Protein
  15. 15. Immunofluorescence
  16. 16. Immunofluorescence
  17. 17. Immunohistochemistry
  18. 18. Assessing Specific Proteins ELISA Western Immunofluorescence $1000 / $2000 / Microscope/COST Machine Machine Core Facility cost of the antibodies $200-400CONVENIENCE Minutes to hours One-two days Two days Localization, Nonspecific, Specificity,COMMENTS duplex, triplex Less information insight re: processing Realtime
  19. 19. Protein, DNA, and mRNA Arrays
  20. 20. Tissue Arrays
  21. 21. Tissue Arrays Histone deacetylase (HDAC4) is highly expressed in the brain and cardiac muscle Liu, et al., MBC 2006
  22. 22. Conclusions • Which Molecular Biology methods you choose depends on one’s specific goals • The choice of method may be guided by the resources and skills available, and the target audience. • The most interesting assays fuse the “tried-and-true” with the “newest-and-greatest”, to answer the most interesting questions. Kao Lab websitehttp://www.xrt.upenn.edu/radiation_biology/Kao_Research.html Mama Jis Molecular Kitchen http://lifesciences.asu.edu/resources/mamajis/index.html

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