Abstract
The Fast Micromethod is a rapid and convenient microplate procedure for the determination of DNA integrity in cell suspensions and in solid tissues. The procedure is based on the ability of fluorochromes to preferentially interact with double-stranded DNA in alkaline conditions. Rapid sample lysis is followed by denaturation at high pH during 15 min. Only 30 ng of DNA from cell suspensions or tissue homogenates per single well are required for the analyses. The whole analysis is performed within 3 h or less (for one 96-well microplate).
The Fast Micromethod is broadly used in biology and medicine. Its applications range from environmental pollution tests in marine invertebrates to the analysis of biopsy samples in cancer patients to detect DNA alterations caused by irradiation or chemotherapy.
The procedure presented here describes the Fast Micromethod applied for the determination of DNA integrity in cell suspensions (HeLa cells) and solid tissues (mussel gills).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Müller WEG, Batel R, Zahn R et al (1997) Mikro-methode zur Schnellbestimmung von DNA-Schäden und deren Reparatur unter Verwendungs des Fluoreszenz farbstoffs PicoGreen und ihre Anwendung. Deutche Patenteamt Muenchen. No. 19724781.4-41
Batel R, Jaksić Ž, Bihari N et al (1999) A microplate assay for DNA damage determination (Fast Micromethod) in cell suspensions and solid tissues. Anal Biochem 270:195–200
Bihari N, Batel R, Jakšić Ž et al (2002) Comparison between the comet assay and Fast Micromethod® for measuring DNA damage in HeLa cells. Croatica Chem Acta 75:793–804
Schröder HC, Batel R, Schwertner H et al (2006) Fast Micromethod DNA single-strand-break assay. In: Henderson DS (ed) DNA repair protocols: mammalian systems. Humana Press Inc, Totowa
Jakšić Ž (2008) Assessing the Fast Micromethod as a tool in DNA integrity measurement. In: Souta M, Shouta N (eds) Progress in DNA damage research. Nova Publishers, New York
Schröder HC, Chauvin C, Lauenroth S et al (1998) Comparison of frequency of apoptosis in X-irradiated human peripheral blood mononuclear cells from donors of various ages. In: Schröder HC, Müller WEG (eds) Development of methods for determination of radiosensitivity. Akademie Gemeinnutziger Wissenschaften, Mainz
Elmendorff-Dreikorn K, Chauvin C, Muller WEG et al (1998) Comparison of frequency of apoptosis in X-irradiated human peripheral blood mononuclear cells from donors of various ages. In: Schröder HC, Müller WEG (eds) Development of methods for determination of radiosensitivity. Akademie Gemeinnutziger Wissenschaften, Mainz
Schröder HC, Batel R, Lauenroth S et al (1999) Induction of DNA damage and expression of heat shock protein HSP70 by polychlorinated biphenyls in the marine sponge Suberites domuncula Olivi. J Exp Mar Biol Ecol 233:285–300
Schröder HC, Hassanein HMA, Lauenroth S et al (1999) Induction of DNA strand breaks and expression of HSP70 and GRP78 homolog by cadmium in the marine sponge Suberites domuncula. Arch Environ Contam Toxicol 36:47–55
Batel R, Fafandjel M, Blumbach B et al (1998) Expression of the human XPB/ERCC-3 excision repair gene-homolog in the sponge Geodia cydonium after exposure to ultraviolet radiation. Mutat Res 409:123–133
Efremova SM, Margulis BA, Guzhova IV et al (2002) Heat shock protein Hsp70 expression and DNA damage in Baikalian sponges exposed to model pollutants and wastewater from Baikalsk Pulp and Paper Plant. Aquat Toxicol 57:267–280
Bihari N, Hamer B, Jakšić Ž et al (2002) Application of alkaline elution, Fast Micromethod and flow cytometry in detection of marine contamination. Cell Mol Biol 48(4):373–377
Bihari N, Mičić M, Fafanđel M et al (2004) Testing quality of sea water from the Adriatic coast of Croatia with toxicity, genotoxicity and DNA integrity tests. Croatica Chem Acta 45(1):75–81
Jakšić Ž, Batel R, Bihari N et al (2005) Adriatic coast as a microcosm for global genotoxic marine contamination—a long-term field study. Mar Pollut Bull 50:1314–1327
Bihari N, Fafanđel M, Jakšić Ž et al (2005) Spatial distribution of DNA integrity in mussels, Mytilusgalloprovincialis, from the Adriatic sea, Croatia. Bull Environ Contam Toxicol 75:845–850
Schröder HC, Batel R, Hassanein HMA et al (2000) Correlation between the level of the potential biomarker, heatshock protein, and the occurrence of DNA damage in the dab, Limanda limanda: a field study in the North Sea and the English Channel. Mar Environ Res 49:201–215
Rye HS, Dabora JM, Quesada MA et al (1993) Fluorometric assay using dimeric dyes for double and single-stranded DNA and RNA with picogram sensitivity. Anal Biochem 208:144–150
Jakšić Ž, Batel R (2003) DNA integrity determination in marine invertebrates by Fast Micromethod®. Aquat Toxicol 65:361–376
Cosa G, Foscaneanu KS, McLean JRN et al (2001) Photophysical properties of fluorescent DNA—dyes bound to single- and double-stranded DNA in aqueous buffered solution. Photochem Photobiol 73:585–599
Meyen RE, Jenkins WT (1983) Variation in normal and tumor tissue sensitivity of mice to ionising radiation induced DNA strand breaks in vivo. Cancer Res 43:5668–5673
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Bihari, N. (2017). Fast Micromethod: Determination of DNA Integrity in Cell Suspensions and in Solid Tissues. In: Didenko, V. (eds) Fast Detection of DNA Damage. Methods in Molecular Biology, vol 1644. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7187-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7187-9_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7185-5
Online ISBN: 978-1-4939-7187-9
eBook Packages: Springer Protocols