radiobio 2006
TRANSCRIPT
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2005 Cancer Biology Course:2005 Cancer Biology Course:Methods in Molecular BiologyMethods in Molecular Biology
Gary D. Kao, M.D., Ph.D.Gary D. Kao, M.D., Ph.D.Department of Radiation OncologyDepartment of Radiation Oncology
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“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
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“Standard Molecular Assays”
DNA - Southern Analysis (blotting)Polymerase Chain Reaction (PCR)Restriction/ linkage (allelic) analyses
RNA - Northern blottingRT (reverse transcriptase)-PCRReal-time PCRMicroarray “Gene array”
Protein – ELISAWestern blottingImmunoprecipitation\ HPLC Immuno-fluorescence\ -histochemistry
Cells – Green fluorescent protein (GFP)-taggingImmunofluorescenceImmunohistochemistry
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Southern (DNA) and Northern (RNA) Analysis
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The Power of PCR:“The Case of the Harmful Hamburger”
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Polymerase Chain Reaction (PCRT) & RT-PCR: DNA & RNA
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Polymerase Chain Reaction (PCRT) & RT-PCR: DNA & RNA
Agarose gel, EtBr -stained
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• 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
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Real-Time PCR
Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at
each PCR cycle (in real time) as opposed to the endpoint detection
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Using the PCR EquationUsing the PCR Equation
Xn = X0(1 + E)n
Xn = PCR product after cycle nX0 = initial copy numberE = amplification efficiencyn = cycle number
Xn
XX00
cycle number
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Southern/ Northern blotting PCR Real-time PCR Microarray
COST
CONVENIENCE
CONCERNS
minimal
$3000 /Machine
$30,000 /Machine
$2000 per expt.
Core Facility
$200 per expt.$20 per expt.
$2000 /Gel box
Radioactive!Takes daysto weeks
easy! Demanding easy!
Radioactive! Most quantitativeneed PCR/ Northernsto confirm!
Semi-quantitative
Assessing DNA & RNA
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Enzyme-Linked Immunosorbent Assay (ELISA)
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Transfer to a Membrane SDS-PAGE Gel
Western blotting: Protein
Antibody localization –therefore Protein - detected by chemilluminescence
Membrane is probed with antibody specific for protein ofinterest
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Western blotting: Protein
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Immunofluorescence
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Immunofluorescence
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Immunohistochemistry
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ELISA Western Immunofluorescence
COST
CONVENIENCE
COMMENTS
$1000 /Machine
$2000 /Machine
Microscope/Core Facility
cost of the antibodies $200-400
Minutes to hours One-two days Two days
Specificity, insight re: processing
Localization, duplex, triplexRealtime
Nonspecific,Less information
Assessing Specific Proteins
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Protein, DNA, and mRNA Arrays
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Tissue Arrays
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Tissue Arrays
Histone deacetylase (HDAC4) ishighly expressed in the brain andcardiac muscle
Liu, et al., MBC 2006
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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.
http://lifesciences.asu.edu/resources/mamajis/index.htmlMama Ji's Molecular Kitchen
http://www.xrt.upenn.edu/radiation_biology/Kao_Research.htmlKao Lab website