Abstract
Conventional DNA ladder assay has certain shortcomings such as loss of DNA fragments during sample processing, involvement of multiple steps and requirement of expensive reagents. The present study demonstrates a rapid, easy-to-perform cost-effective method for detection of apoptotic DNA fragments with considerable improvement in the sensitivity by avoiding loss of DNA fragments. It involves a few minutes of procedure involving direct lysis of cells with dimethyl sulphoxide (DMSO), brief vortexing, addition of 2% SDS–TE buffer, and a single step of centrifugation. This cost- and time-efficient method reduces the assay time considerably and can be used for a large number of samples with excellent sensitivity.
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References
Arakawa T, Kita Y, Timasheff SN (2007) Protein precipitation and denaturation by dimethyl sulfoxide. Biophys Chem 131:62–70
Basnakian AG, James SJ (1994) A rapid and sensitive assay for detection of DNA fragmentation during early phases of apoptosis. Nucleic Acids Res 22:2714–2715
Chandna S (2004) Single-cell gel electrophoresis assay monitors precise kinetics of DNA fragmentation induced during programmed cell death. Cytometry Part A 61A:127–133
Chandna S, Dwarakanath BS, Seth RK, Khaitan D, Adhikari JS, Jain V (2004) Radiation responses of Sf9, a highly radioresistant lepidopteran insect cell line. Int J Radiat Biol 80:301–315
Compton MM (1992) A biochemical hallmark of apoptosis: intranucleosomal degradation of the genome. Cancer Metast Rev 11:105–119
Czene S, Testa E, Nygren J, Belyaev I, Harms-Ringdahl MH (2002) DNA fragmentation and morphological changes in apoptotic human lymphocytes. Biochem Biophys Res Commun 294:872–878
Di L, Kerns EH (2006) Biological assay challenges from compound solubility: strategies for bioassay optimization. Drug Discov Today 11:446–451
Gong J, Traganos F, Darzynkiewicz Z (1994) A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem 218:314–319
Hermann BG, Frischauf AM (1987) Isolation of genomic DNA. Methods Enzymol (US) 152:180–183
Kang J, Lee MS, Gorenstein DG (2005) The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J Biochem Biophys Methods 64:147–151
Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257
Kumarswamy R, Seth RK, Dwarakanath BS, Chandna S (2009) Mitochondrial regulation of insect cell apoptosis: evidence for permeability transition pore-independent cytochrome-c release in the Lepidopteran Sf9 cells. Int J Biochem Cell Biol 41:1430–1440
Lamm GM, Steinlein P, Cotton M, Christofori G (1997) A rapid, quantitative and inexpensive method for detecting apoptosis by flow cytometry in transiently transfected cells. Nucleic Acids Res 25:4855–4857
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–280
Otsuki Y (2000) Various methods of apoptosis detection. Acta Histochem Cytochem 33:235–241
Pandey S, Walker PR, Sikorska M (1994) Separate pools of endonuclease activity are responsible for inter-nucleosomal and high-molecular-mass DNA fragmentation during apoptosis. Biochem cell Biol (biochimie et biologie cellulaire) 72:625–629
Rosl F (1992) A simple and rapid method for detection of apoptosis in human cells. Nucleic Acids Res 20:5243
Saraste A, Pulkki K (2000) Morphological and biochemical hallmarks of apoptosis. Cardiovasc Res 45:528–537
Sgonc R, Guber J (1998) Apoptosis detection: an overview. Exp Gerontol 33:525–533
Singh NP (2000) A simple method for accurate estimation of apoptotic cells. Exp Cell Res 256:328–337
Suman S, Khaitan D, Pati U, Seth RK, Chandna S (2009) Stress response of a p53 homologue in the radioresistant Sf9 insect cells. Int J Radiat Biol 85:238–249
Tanuma S, Shiokawa D, Tanimoto Y, Ikekita M, Sakagami H, Takada M, Fukuda S, Kochi M (1993) Benzylideneascorbate induces apoptosis in L029 tumor cells. Biochem Biophys Res Commun 15:29–35
Telford WG, King LE, Fraker PJ (1991) Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow-cytometry. Cell Prolif 24:447–459
Telford WG, King LE, Fraker PJ (1992) Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 13:137–143
Umansky SR, Korol BR, Nelipaovich PA (1981) In vivo DNA degradation in the thymocytes of gamma-irradiated or hydrocortisone-treated rats. Biochim Biophys Acta 655:281–290
Wang G, Gong Y, Burczynski FJ, Hasinoff BB (2008) Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay. Free Radic Res 42:435–441
Willingham MC (1999) Cytochemical methods for the detection of apoptosis. J Histochem Cytochem 47:1101–1109
Zhuang Z, Fei F, Chen Y, Jin W (2008) Suberoyl bis-hydroxamic acid induces p53-dependent apoptosis of MCF-7 breast cancer cells. Acta Pharmacol Sin 29:1459–1466
Acknowledgment
This work is an offshoot from the routine cellular radiobiology studies being carried out under project INM-311.1.5 funded by DRDO, Government of India. Constant support received in our studies from Dr. R. P. Tripathi, Director INMAS, is duly acknowledged.
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Suman, S., Pandey, A. & Chandna, S. An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis. Cytotechnology 64, 9–14 (2012). https://doi.org/10.1007/s10616-011-9395-0
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DOI: https://doi.org/10.1007/s10616-011-9395-0