Abstract
This chapter describes step-by-step a fast protocol to obtain a crude extract of genomic DNA from formalin-fixed paraffin-embedded (FFPE) tissue for PCR analysis. The time required for the procedure is approximately 1.5 h with an overnight incubation. This method may be applied for either entire tissue sections or microdissected FFPE tissues. The protocol is based on the method described by Higuchi (1989, Stockton Press, New York), with modifications concerning the deparaffinization steps and the tissue recovery. For the described method, a tissue section (3–5 µm thickness) containing about 1 cm2 area is required for the basic protocol. Additional information is given when dealing with smaller tissue samples resulting from microdissections. After cutting, the tissue is deparaffinized, followed by proteolytic treatment with Proteinase K. The DNA can be quantitated photometrically and used directly for PCR analysis.
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Notes
- 1.
Alternatively, the ready-to-use Proteinase K solution (Qiagen 19133) can be used; it has a concentration of about 20 mg/ml (store in aliquots at +4°C).
- 2.
Clean pipettes with DNase Away™ to avoid DNase and DNA contamination. Alternatively, it is possible to clean pipettes by first using mild detergent containing aqueous solutions, followed by application of antiseptic alcohol solution (e.g., 70% (v/v) ethanol) or another disinfectant and then leaving them under UV light for at least 10 min.
- 3.
Depending on the Thermomixer, smaller microreaction tubes may be used.
- 4.
The concentration of dsDNA expressed in μg/μl is obtained as follows: [DNA] = A260 × dilution factor × 50 × 10−3. A clean DNA preparation should have a A260/A280 ratio of 1.5–2. This ratio is decreased by the presence of proteins, oligo- and polysaccharides. Concentration estimation can be also affected by phenol contamination, as phenol absorbs strongly at 260 nm and therefore can mimic higher DNA yield and purity.
Reference
Higuchi R (1989) Simple and rapid preparation of samples for PCR. PCR technology; principles and applications for DNA amplification. Stockton, New York
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© 2011 Springer-Verlag Berlin Heidelberg
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Hlubek, F., Jung, A. (2011). Fast Protocol for DNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_9
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DOI: https://doi.org/10.1007/978-3-642-17890-0_9
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