2010 Volume No 20 – pages 38-44
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Title: Ceramic materials lead to underestimated DNA quantifications: a method for reliable measurements |
Author: E Piccinini, N Sadr, I Martin |
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Address: Institute for Surgical Research and Hospital Management, University Hospital Basel, Hebelstrasse 20, ZLF, Room 405, CH-4031 Basel, Switzerland |
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E-mail: imartin at uhbs.ch |
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Key Words: DNA extraction, DNA quantification, ceramic, tissue engineering |
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Publication date: July 22nd 2010 |
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Abstract: In the context of investigating cell-material interactions or of material-guided generation of tissues, DNA quantification represents an elective method to precisely assess the number of cells attached or embedded within different substrates. Nonetheless, nucleic acids are known to electrostatically bind to ceramics, a class of materials commonly employed in orthopaedic implants and bone tissue engineering scaffolds. This phenomenon is expected to lead to a relevant underestimation of the DNA amount, resulting in erroneous experimental readouts. The present work aims at (i) investigating the effects of DNA-ceramic bond occurrence on DNA quantification, and (ii) developing a method to reliably extract and accurately quantify DNA in ceramic-containing specimens. A cell-free model was adopted to study DNA-ceramic binding, highlighting an evident DNA loss (up to 90%) over a wide range of DNA/ceramic ratios (w/w). A phosphate buffer-based (800mM) enzymatic extraction protocol was developed and its efficacy in terms of reliable DNA extraction and measurement was confirmed with commonly used fluorometric assays, for various ceramic substrates. The proposed buffered DNA extraction technique was validated in a cell-based experiment showing 95% DNA retrieval in a cell seeding experiment, demonstrating a 3.5-fold increase in measured DNA amount as compared to a conventional enzymatic extraction protocol. In conclusion, the proposed phosphate buffer method consistently improves the DNA extraction process assuring unbiased analysis of samples and allowing accurate and sensitive cell number quantification on ceramic containing substrates.
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