1. Dr. Sabine A.S. Langie hCOMET-COST training school
Tips & tricks
Statistical analysis of comet-based
in vitro repair data
Dr. Sabine A.S. Langie
hCOMET-COST training school

2. Following the decrease of DNA damage over time.

3. In vitro assay
Exposure to Ro plus light or KBrO3 (BER) UV or BPDE (NER)
Tissue
Substrate cells embedded in gel
Protein/enzyme extract
Nucleoids containing 8-oxodG lesions
Cell lysis - +
Incubation at 37°C
Denaturation / electrophoresis
Analyze comets

4. Combining Cellular repair and In vitro repair
Multiple values
At least 2
1 value
How to convert?
Average % per time
difference
Slope (linear?)
Velocity of DNA damage removal
Half-life (T1/2)
Do we take initial damage at t=1
into account?

5. Comparing Cellular repair and In vitro repair

6. In vitro assay
Exposure to Ro plus light or KBrO3 (BER) UV or BPDE (NER)
Tissue
Substrate cells embedded in gel
Protein/enzyme extract
Nucleoids containing 8-oxodG lesions
Cell lysis - +
Incubation at 37°C
Denaturation / electrophoresis
Analyze comets

7. Repair capacity = (D-C)-B (after correction for background)
noRo/PBS
Buffer A
noRo/PBS
Extract C
Ro+light
Extract D
In the next slide I’ll give you a theoretical figure of some data
The tail moments are represented as the mean +- SD
The first bar (A) represents the average TM of the unexposed nuclei  background
The next bar represents nuclei only exposed to the vehicle control and incubated with the protein extracts
 they give an indication about the recognition of the endogenous DNA damage
The last bar show the tail moment of nuclei exposed to BPDE and Extract  these values give an idea about the specific recognition of BPDE adducts
The second bar gives the TM of the nuclei exposed to BPDE alone
From these data the repair capacity can be calculated, resulting in a value that will tell us something about the specific recognition of BPDE-DNA adducts by the extracts
Ro+light
Buffer B

8. The ever lasting discussion – TM or TI???

9. Rational for using tail moment ( represent BPDE-DNA adducts)
TM or TI??? – Our opinion
Rational for using tail moment ( represent BPDE-DNA adducts)

10. The ever lasting discussion – TM or TI???

11. The ever lasting discussion – TM or TI???
Correlation BPDE adduct removal vs. NER assay
P = 0,005
P = 0,041

12. Data normalisation
How to correct for inter-assay variations?

13. Using the exposure of substrate as controle?

14. Excel templates

15. Using Ro or FPG data to correct?
Data normalization

16. Using Ro or FPG data to correct?

17. Include buffer controls  to correct for any background damage (recognition)
Include exposure controls  to compare experiments with different batches of substrate cells
Include (positive) assay controls (e.g. FPG)  to correct for inter-assay variation when using the same batch of substrate cells
When no suitable enzyme is available as assay control (e.g. in case of BPDE-NER assay)  use a pooled sample in each assay to normalize the data