Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assay
Abstract The comet assay is an electrophoretic technique used to assess DNA damage, as a marker of genotoxicity and oxidative stress, in tissues and biological samples including peripheral blood mononuclear cells (PBMCs) and whole blood (WB). Although numerous studies are performed on stored samples...
Main Authors: | , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Nature Publishing Group
2021-01-01
|
Series: | Scientific Reports |
Online Access: | https://doi.org/10.1038/s41598-020-79670-8 |
id |
doaj-1c990feaff6b4e1889d1f445544b1b64 |
---|---|
record_format |
Article |
spelling |
doaj-1c990feaff6b4e1889d1f445544b1b642021-01-17T12:33:04ZengNature Publishing GroupScientific Reports2045-23222021-01-0111111110.1038/s41598-020-79670-8Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assayMirko Marino0Letizia Gigliotti1Peter Møller2Patrizia Riso3Marisa Porrini4Cristian Del Bo5Division of Human Nutrition, Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di MilanoDivision of Human Nutrition, Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di MilanoDepartment of Public Health, Section of Environmental Health, University of CopenhagenDivision of Human Nutrition, Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di MilanoDivision of Human Nutrition, Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di MilanoDivision of Human Nutrition, Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di MilanoAbstract The comet assay is an electrophoretic technique used to assess DNA damage, as a marker of genotoxicity and oxidative stress, in tissues and biological samples including peripheral blood mononuclear cells (PBMCs) and whole blood (WB). Although numerous studies are performed on stored samples, the impact of cryopreservation on artifactual formation of DNA damage is not widely considered. The present study aims to evaluate the impact of storage at different time-points on the levels of strand breaks (SBs) and formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites in isolated PBMCs and WB. Samples were collected, aliquoted and stored at − 80 °C. DNA damage was analyzed on fresh samples, and subsequently on frozen samples every 2 months up to a year. Results have shown no changes in DNA damage in samples of PBMCs and WB stored for up to 4 months, while a significant increase in SBs and Fpg-sensitive sites was documented starting from 6-month up to 12-month storage of both the samples. In addition, fresh and frozen WB showed higher basal levels of DNA damage compared to PBMCs. In conclusion, WB samples show high levels of DNA damage compared to PBMCs. One-year of storage increased the levels of SBs and Fpg-sensitive sites especially in the WB samples. Based on these findings, the use of short storage times and PBMCs should be preferred because of low background level of DNA damage in the comet assay.https://doi.org/10.1038/s41598-020-79670-8 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Mirko Marino Letizia Gigliotti Peter Møller Patrizia Riso Marisa Porrini Cristian Del Bo |
spellingShingle |
Mirko Marino Letizia Gigliotti Peter Møller Patrizia Riso Marisa Porrini Cristian Del Bo Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assay Scientific Reports |
author_facet |
Mirko Marino Letizia Gigliotti Peter Møller Patrizia Riso Marisa Porrini Cristian Del Bo |
author_sort |
Mirko Marino |
title |
Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assay |
title_short |
Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assay |
title_full |
Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assay |
title_fullStr |
Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assay |
title_full_unstemmed |
Impact of 12-month cryopreservation on endogenous DNA damage in whole blood and isolated mononuclear cells evaluated by the comet assay |
title_sort |
impact of 12-month cryopreservation on endogenous dna damage in whole blood and isolated mononuclear cells evaluated by the comet assay |
publisher |
Nature Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2021-01-01 |
description |
Abstract The comet assay is an electrophoretic technique used to assess DNA damage, as a marker of genotoxicity and oxidative stress, in tissues and biological samples including peripheral blood mononuclear cells (PBMCs) and whole blood (WB). Although numerous studies are performed on stored samples, the impact of cryopreservation on artifactual formation of DNA damage is not widely considered. The present study aims to evaluate the impact of storage at different time-points on the levels of strand breaks (SBs) and formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites in isolated PBMCs and WB. Samples were collected, aliquoted and stored at − 80 °C. DNA damage was analyzed on fresh samples, and subsequently on frozen samples every 2 months up to a year. Results have shown no changes in DNA damage in samples of PBMCs and WB stored for up to 4 months, while a significant increase in SBs and Fpg-sensitive sites was documented starting from 6-month up to 12-month storage of both the samples. In addition, fresh and frozen WB showed higher basal levels of DNA damage compared to PBMCs. In conclusion, WB samples show high levels of DNA damage compared to PBMCs. One-year of storage increased the levels of SBs and Fpg-sensitive sites especially in the WB samples. Based on these findings, the use of short storage times and PBMCs should be preferred because of low background level of DNA damage in the comet assay. |
url |
https://doi.org/10.1038/s41598-020-79670-8 |
work_keys_str_mv |
AT mirkomarino impactof12monthcryopreservationonendogenousdnadamageinwholebloodandisolatedmononuclearcellsevaluatedbythecometassay AT letiziagigliotti impactof12monthcryopreservationonendogenousdnadamageinwholebloodandisolatedmononuclearcellsevaluatedbythecometassay AT petermøller impactof12monthcryopreservationonendogenousdnadamageinwholebloodandisolatedmononuclearcellsevaluatedbythecometassay AT patriziariso impactof12monthcryopreservationonendogenousdnadamageinwholebloodandisolatedmononuclearcellsevaluatedbythecometassay AT marisaporrini impactof12monthcryopreservationonendogenousdnadamageinwholebloodandisolatedmononuclearcellsevaluatedbythecometassay AT cristiandelbo impactof12monthcryopreservationonendogenousdnadamageinwholebloodandisolatedmononuclearcellsevaluatedbythecometassay |
_version_ |
1714941913480560640 |