Single cell gel electrophoresis (SCGE), also known as comet assay is a widely used method to detect DNA damage. Its use is nonetheless subjected to some pitfalls, due to differences in experimental set-up, to operator-dependent variability and to quantification of the comets, which is usually accomplished by visual scoring or by image-analysis software. Biological variability in the extent of DNA damage must be taken into account particularly regarding in vivo studies. In the present paper we propose an improved methodology where major features are: a) cryopreservation of lymphocytes collected at different time points and simultaneous analysis in a single run; b) use of an internal control on each slide; c) development of a custom-made software with semi - automated image analysis in order to overcome operator dependent variability. Cryopreservation was accomplished by storing lymphocytes in liquid nitrogen in a solution commonly used for preserving vital cells to be reinfused. We found that this procedure did not alter DNA after 2 and 4 months of storage. The use of quality control from a batch of aliquoted lymphocytes from a healthy donor on each slide, enabled to highlight possible experimental anomalies as well as verify inter-experimental variability. Moreover, by using a newly developed software able to automatically recognise comets we minimised operator-dependent variability in the scoring process. This improved methodology is proposed for longitudinal in vivo studies and in the present work its application made it possible to assess a significant increase of DNA in pediatric Down Syndrome patients compared to healthy controls of the same age.

Assessment of DNA damage in Down syndrome patients by means of a new, optimised single cell gel electrophoresis technique

TIANO, LUCA;LITTARRU, GIAN PAOLO;GABRIELLI, ORAZIO
2005-01-01

Abstract

Single cell gel electrophoresis (SCGE), also known as comet assay is a widely used method to detect DNA damage. Its use is nonetheless subjected to some pitfalls, due to differences in experimental set-up, to operator-dependent variability and to quantification of the comets, which is usually accomplished by visual scoring or by image-analysis software. Biological variability in the extent of DNA damage must be taken into account particularly regarding in vivo studies. In the present paper we propose an improved methodology where major features are: a) cryopreservation of lymphocytes collected at different time points and simultaneous analysis in a single run; b) use of an internal control on each slide; c) development of a custom-made software with semi - automated image analysis in order to overcome operator dependent variability. Cryopreservation was accomplished by storing lymphocytes in liquid nitrogen in a solution commonly used for preserving vital cells to be reinfused. We found that this procedure did not alter DNA after 2 and 4 months of storage. The use of quality control from a batch of aliquoted lymphocytes from a healthy donor on each slide, enabled to highlight possible experimental anomalies as well as verify inter-experimental variability. Moreover, by using a newly developed software able to automatically recognise comets we minimised operator-dependent variability in the scoring process. This improved methodology is proposed for longitudinal in vivo studies and in the present work its application made it possible to assess a significant increase of DNA in pediatric Down Syndrome patients compared to healthy controls of the same age.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11566/51028
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