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

“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) analyses

RNA - Northern blottingRT (reverse transcriptase)-PCRReal-time PCRMicroarray “Gene array”

Protein – ELISAWestern blottingImmunoprecipitation\ HPLC Immuno-fluorescence\ -histochemistry

Cells – Green fluorescent protein (GFP)-taggingImmunofluorescenceImmunohistochemistry

Southern (DNA) and Northern (RNA) Analysis

The Power of PCR:“The Case of the Harmful 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 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

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

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

Enzyme-Linked Immunosorbent Assay (ELISA)

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

Western blotting: Protein

Immunofluorescence

Immunofluorescence

Immunohistochemistry

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

Protein, DNA, and mRNA Arrays

Tissue Arrays

Tissue Arrays

Histone deacetylase (HDAC4) ishighly expressed in the brain andcardiac 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.

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

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