Affiliations: Institute of Biochemistry, Polytechnic University of
the Marche, Ancona, Italy | Faculty of Engineering, Polytechnic University of the
Marche, Ancona, Italy | Institute of Maternal-Infantile Science, Polytechnic
University of the Marche, Ancona, Italy
Note: [] Address for correspondence: Luca Tiano, Institute of
Biochemistry, Polytechnic University of the Marche, Ancona, Italy. Tel.: +39
0712204394; Fax: +39 0712204398; E-mail: luca.tiano@unicam.it
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.
Keywords: Comet assay, Down Syndrome, DNA damage
Journal: BioFactors, vol. 25, no. 1-4, pp. 187-195, 2005
