radiobio 2006

23
2005 Cancer Biology Course: 2005 Cancer Biology Course: Methods in Molecular Biology Methods in Molecular Biology Gary D. Kao, M.D., Ph.D. Gary D. Kao, M.D., Ph.D. Department of Radiation Oncology Department of Radiation Oncology

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Page 1: Radiobio 2006

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

Page 2: Radiobio 2006

“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

Page 3: Radiobio 2006

“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

Page 4: Radiobio 2006

Southern (DNA) and Northern (RNA) Analysis

Page 5: Radiobio 2006

The Power of PCR:“The Case of the Harmful Hamburger”

Page 6: Radiobio 2006

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

Page 15: Radiobio 2006

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

Page 23: Radiobio 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.

http://lifesciences.asu.edu/resources/mamajis/index.htmlMama Ji's Molecular Kitchen

http://www.xrt.upenn.edu/radiation_biology/Kao_Research.htmlKao Lab website