radiobio

1. 2005 Cancer Biology Course:
Methods in Molecular Biology
Gary D. Kao, M.D., Ph.D.
Department of Radiation Oncology

2. “The progression of scientific enquiry”
DNA - “the blue print for mRNA”
RNA - “regulated or dysregulated expression”
Protein - too much, too little, or mutant proteins
Cancer Cells – proliferating, invasive, metastatic
Animal models - xenografts vs. endogenous tumors
Patients - retrospective vs prospective studies

3. “Standard Molecular Assays”
DNA - Southern Analysis (blotting)
Polymerase Chain Reaction (PCR)
Restriction/ linkage (allelic) analyses
RNA - Northern blotting
RT (reverse transcriptase)-PCR
Real-time PCR
Microarray “Gene array”
Protein – ELISA
Western blotting
Immunoprecipitation HPLC
Immuno-fluorescence -histochemistry
Cells – Green fluorescent protein (GFP)-tagging
Immunofluorescence
Immunohistochemistry

4. Southern (DNA) and Northern (RNA) Analysis

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

6. Polymerase Chain Reaction (PCRT) & RT-PCR: DNA & RNA

7. Polymerase Chain Reaction (PCRT) & RT-PCR: DNA & RNA
Agarose gel, EtBr -stained

8. • 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

9. 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

11. Using the PCR Equation
Xn
Xn = X0(1 + E)n
Xn = PCR product after cycle n
X0 = initial copy number
E = amplification efficiency
n = cycle number X0
cycle number

12. Assessing DNA & RNA
Southern/ Microarray
PCR Real-time PCR
Northern blotting
$2000 / $3000 / $30,000 /
Core Facility
Gel box Machine Machine
COST
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!

13. Enzyme-Linked Immunosorbent Assay (ELISA)

14. 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

15. Western blotting: Protein

16. Immunofluorescence

17. Immunofluorescence

18. Immunohistochemistry

19. Assessing Specific Proteins
ELISA Western Immunofluorescence
$1000 / $2000 / Microscope/
COST
Machine Machine Core Facility
cost of the antibodies $200-400
CONVENIENCE Minutes to hours One-two days Two days
Localization,
Nonspecific, Specificity,
COMMENTS duplex, triplex
Less information insight re: processing
Realtime

20. Protein, DNA, and mRNA Arrays

21. Tissue Arrays

22. Tissue Arrays
Histone deacetylase (HDAC4) is
highly expressed in the brain and
cardiac muscle
Liu, et al., MBC 2006

23. 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 website
http://www.xrt.upenn.edu/radiation_biology/Kao_Research.html
Mama Ji's Molecular Kitchen
http://lifesciences.asu.edu/resources/mamajis/index.html