Novel DNA Staining Method and Processing Technique for the Quantification of Undamaged Double-stranded DNA in Epidermal Tissue Sections by PicoGreen Probe Staining and Microspectrophotometry
Claude E. Gagna 1*, Hon-Reen Kuo 1, Norman J. Chan 1, Eugene J. Mitacek 1, Alla Spivak 1, Tiffany D. Pasquariello 1, Chandrika Balgobin 1, Ruhayna Mukhi 1 and W. Clark Lambert 1
1 Department of Pathology and Laboratory Medicine (CEG,H-RK,WCL) and Department of Medicine (CEG,WCL), New Jersey Medical School, Newark, New Jersey, and School of Health Professions, Behavioral and Life Sciences, New York Institute of Technology, Old Westbury, New York (CEG,NJC,EJM,AS,TDP,CB,RM)
* To whom correspondence should be addressed. E-mail: dr.c.gagna{at}att.net.
Submitted on January 19, 2007
Accepted on 10 May 2007
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Abstract |
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Histotechnological processing of DNA can cause damage to and loss of DNA, and can change its structure. DNA probes have severe tissue-staining limitations. New DNA probes and improved histotechnology are needed to enhance the characterization of fixed tissue-bound DNA. Our team developed a novel DNA staining technique and histotechnological processing procedure that improves tissue-bound DNA retention and the qualification and quantification of intact double-stranded (ds-) B-DNA. We used the ultrasensitive PicoGreen ds-DNA probe for the histochemical characterization of ds-DNA. Fifteen fixatives were examined to determine which were better for preventing DNA denaturation and retaining original DNA content and structures. Our use of a microwave-vacuum oven reduced heating temperatures, shortened heating and processing times, and enhanced fixation. We achieved better qualitative and quantitative results by using superior tissue-acquisition techniques (e.g., reduced prefixation times), and improved histotechnology. We also compared our novel approach with archival tissues, delayed fixation, less sophisticated, conventional histological processing techniques, and by experimenting with preservation of tissue-bound ds-Z-DNA. The results demonstrate that our histotechnological procedure and nucleic acid staining method significantly improve the retention of intact, undamaged ds-DNA which, in turn, allows the investigator to more precisely quantify the content and structures of unaltered undamaged tissue-bound ds-B-DNA.
Key Words:
PicoGreen, DNA quantification, DNA structure, B-DNA, Z-DNA, fixatives, histochemistry, histotechnology, tissue processing, double-stranded DNA, denatured DNA
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