Radiobio 2006
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Transcript of 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