Abstract
This chapter provides a reliable home-made method of obtaining DNA from small specimens of formalin-fixed and paraffin-embedded (FFPE) tissues. The described method is dedicated mainly to microdissected tissues. The method described hereafter is based on a proteolytic digestion and a temperature deactivation of Proteinase K using a point-to-point protocol with notes and references to guide the researcher into the laboratory practice. The DNA extracts obtained by the application of this method are suitable for most of the PCR analyses used in molecular pathology laboratories, ranging from single to complex multiplexed PCRs or B-/T-cell clonality PCR tests, as well as for PCR sequencing.
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Notes
- 1.
The solubilization of Proteinase K in 50% sterile glycerol maintains the solution fluid at −20°C with a better preservation of the enzymatic activity.
- 2.
Clean the pipettes with alcohol or another disinfectant and leave them under the UV lamp for at least 10 min. Alternatively, it is possible to autoclave the pipette depending on the provider instructions.
- 3.
It is possible to bypass the deparaffinization step by adding the digestion buffer directly to the cut sections. In this case, it is better to add a short incubation time of 5 min at 65°C to melt the paraffin rapidly [ 6 ].
- 4.
When working with xylene, avoid breathing fumes; it is better to perform the deparaffinization step under a fume hood.
- 5.
Wear gloves when isolating and handling DNA to minimize the contamination with exogenous nucleases. Use autoclaved pipette tips and 1.5 ml microcentrifuge tubes.
- 6.
Xylene is harmful; the wasted xylene must be collected in a chemical waste container and discharged according to the local hazardous chemical disposal procedures.
- 7.
If the pellet is firmly lodged at the bottom of the tube, it is possible to dislodge it in the digestion buffer using a sterile toothpick.
- 8.
Long digestion (at least 48 h) increases the yield of the DNA.
- 9.
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.
References
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Bonin, S., Groenen, P.J.T.A., Halbwedl, I., Popper, H.H. (2011). DNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues (FFPE) (from Small Fragments of Tissues or Microdissected Cells). In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_8
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DOI: https://doi.org/10.1007/978-3-642-17890-0_8
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