A modified fluorimetric neutral filter elution method for analyzing radiation-induced double strand break and repair

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Abstract

Neutral filter elution assay is one of the methods used for detection of DNA double strand breaks (DSBs). However, it is laborious, expensive, and hazardous (radiolabeled precursors for DSB detection and scintillation counter for quantification), making it a less preferred method for DSB detection. In the present study, an attempt was made to improve the existing neutral filter elution assay by making use of fluorescent dye (PicoGreen) and microfiltration assembly for eluting the fragmented DNA, thereby reducing the cost and time required for the assay. We studied the effect of dye dilution, pH conditions, and cell number as a part of method standardization. X-ray dose-response and repair kinetics in lymphocytes as well as cell lines were studied for validating the sensitivity of the assay. A linear dose-response relationship for DSBs was observed at a cell number of 4 × 105 cells, a dye dilution of 500-fold, and at pH 10. Repair kinetics revealed a time-dependent repair of DSBs up to 360 min of posttreatment, indicating its usefulness in DSB repair studies. In conclusion, the present modified method is more efficient (in terms of cell number), cost effective, less time-consuming, and less hazardous compared to the existing method.

Original languageEnglish
Pages (from-to)287-293
Number of pages7
JournalAnalytical Biochemistry
Volume414
Issue number2
DOIs
Publication statusPublished - 15-07-2011

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Assays
Repair
Radiation
Cell Count
Dilution
Coloring Agents
Scintillation counters
Kinetics
Microfiltration
Lymphocytes
DNA
Costs and Cost Analysis
Scintillation Counting
Fluorescent Dyes
Double-Stranded DNA Breaks
Standardization
Dosimetry
Costs
Cells
X rays

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "A modified fluorimetric neutral filter elution method for analyzing radiation-induced double strand break and repair",
abstract = "Neutral filter elution assay is one of the methods used for detection of DNA double strand breaks (DSBs). However, it is laborious, expensive, and hazardous (radiolabeled precursors for DSB detection and scintillation counter for quantification), making it a less preferred method for DSB detection. In the present study, an attempt was made to improve the existing neutral filter elution assay by making use of fluorescent dye (PicoGreen) and microfiltration assembly for eluting the fragmented DNA, thereby reducing the cost and time required for the assay. We studied the effect of dye dilution, pH conditions, and cell number as a part of method standardization. X-ray dose-response and repair kinetics in lymphocytes as well as cell lines were studied for validating the sensitivity of the assay. A linear dose-response relationship for DSBs was observed at a cell number of 4 × 105 cells, a dye dilution of 500-fold, and at pH 10. Repair kinetics revealed a time-dependent repair of DSBs up to 360 min of posttreatment, indicating its usefulness in DSB repair studies. In conclusion, the present modified method is more efficient (in terms of cell number), cost effective, less time-consuming, and less hazardous compared to the existing method.",
author = "Goutham, {Venkatesh H.} and Kamalesh, {Mumbrekar D.} and Guruprasad, {Parashiva K.} and Vadhiraja, {Manjunath B.} and Kapaettu Satyamoorthy and {Rao Bola Satish}, Sadashiva",
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AU - Kamalesh, Mumbrekar D.

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AU - Vadhiraja, Manjunath B.

AU - Satyamoorthy, Kapaettu

AU - Rao Bola Satish, Sadashiva

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AB - Neutral filter elution assay is one of the methods used for detection of DNA double strand breaks (DSBs). However, it is laborious, expensive, and hazardous (radiolabeled precursors for DSB detection and scintillation counter for quantification), making it a less preferred method for DSB detection. In the present study, an attempt was made to improve the existing neutral filter elution assay by making use of fluorescent dye (PicoGreen) and microfiltration assembly for eluting the fragmented DNA, thereby reducing the cost and time required for the assay. We studied the effect of dye dilution, pH conditions, and cell number as a part of method standardization. X-ray dose-response and repair kinetics in lymphocytes as well as cell lines were studied for validating the sensitivity of the assay. A linear dose-response relationship for DSBs was observed at a cell number of 4 × 105 cells, a dye dilution of 500-fold, and at pH 10. Repair kinetics revealed a time-dependent repair of DSBs up to 360 min of posttreatment, indicating its usefulness in DSB repair studies. In conclusion, the present modified method is more efficient (in terms of cell number), cost effective, less time-consuming, and less hazardous compared to the existing method.

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