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Radiobio 2006

Radiobio 2006







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    Radiobio 2006 Radiobio 2006 Presentation Transcript

    • 2005 Cancer Biology Course:Methods in Molecular Biology Gary D. Kao, M.D., Ph.D. Department of Radiation Oncology
    • “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
    • “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
    • Southern (DNA) and Northern (RNA) Analysis
    • The Power of PCR:“The Case of theHarmful Hamburger”
    • Polymerase Chain Reaction (PCRT) & RT-PCR: DNA & RNA
    • Polymerase Chain Reaction (PCRT) & RT-PCR: DNA & RNA Agarose gel, EtBr -stained
    • • 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
    • 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
    • Using the PCR Equation XnXn = X0(1 + E)nXn = PCR product after cycle nX0 = initial copy numberE = amplification efficiencyn = cycle number X0 cycle number
    • 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!
    • Enzyme-Linked Immunosorbent Assay (ELISA)
    • 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
    • Western blotting: Protein
    • Immunofluorescence
    • Immunofluorescence
    • Immunohistochemistry
    • 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
    • Protein, DNA, and mRNA Arrays
    • Tissue Arrays
    • Tissue Arrays Histone deacetylase (HDAC4) is highly expressed in the brain and cardiac muscle Liu, et al., MBC 2006
    • 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