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

Following the decrease of DNA damage over time.

In vitro assay Exposure to Ro 19-8022 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

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?

Comparing Cellular repair and In vitro repair

In vitro assay Exposure to Ro 19-8022 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

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

The ever lasting discussion – TM or TI???

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

The ever lasting discussion – TM or TI???

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

Data normalisation How to correct for inter-assay variations?

Using the exposure of substrate as controle?

Excel templates

Using Ro or FPG data to correct? Data normalization

Using Ro or FPG data to correct?

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