DNA Extraction from Formalin-Fixed Paraffin-Embedded (FFPE) Tissues

Abstract

The aim of this chapter is to provide a reliable method of obtaining DNA from formalin-fixed and paraffin-embedded (FFPE) tissue specimens. The application of this method allows the extraction of DNA from specimens of both biopsy and autopsy origin. The method described here is based on proteolytic digestion, phenol purification, and alcohol precipitation, 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.

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction and Purpose

This protocol provides a method of obtaining DNA suitable for PCR analyses from formalin-fixed and paraffin-embedded (FFPE) tissue specimens [1–4], even of autopsy origin [5, 6]. This procedure is based mainly on deparaffinization of tissues (as optional step) and a proteolytic digestion with Proteinase K. The proteolysis step is fundamental to degrade proteins and generate pure DNA. The time required for the whole procedure is 4 days.

Commercial kits are also available for DNA extraction from FFPE; some of these are specifically dedicated to archive tissues (i.e., QIAamp DNA FFPE Tissue Kit). However, some minor modifications could be useful to achieve better results depending on the type of tissues; requirements for the specific molecular test should be taken into consideration [7].

2 Protocol

2.1 Reagents

Note: Reagents from specific companies are reported here, but similar reagents from other providers could be used:

• Xylene (Fluka or Sigma-Aldrich)

• Absolute, 90% and 70% Ethanol (Sigma)

• 20 mg/ml Proteinase K (stock solution): (Sigma P2303) Dissolve 100 mg of Proteinase K in 5 ml of autoclaved 50% glycerol diluted in sterile H₂O.¹ Store at −20°C. The stock solution of 20 mg/ml should be diluted to a final concentration of 1 mg/ml Proteinase K in digestion buffer

• 10× Digestion Buffer ² (stock solution): 500 mM Tris HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 5% Tween 20

• 1× Digestion Buffer: 50 mM Tris HCl pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.5% Tween 20. Complete the solution with Proteinase K (1 mg/ml  =  final concentration) just before use

• Phenol-Tris buffered pH 8 ³ /CHCl ₃ 50:50: Mix 1 part of buffered phenol with 1 part of chloroform. Top the organic phase with 1× TE buffer (about 1 cm height) and allow the phase to separate. Store at 4°C in a light-tight bottle

• Phenol-Tris buffered pH 8/CHCl ₃ – isoamyl alcohol 50:49:1: Mix 48 ml of Chloroform with 2 ml of isoamyl alcohol. Mix 1 part of buffered phenol with 1 part of chloroform–isoamyl alcohol. Top the organic phase with 1× TE buffer (about 1 cm high) and allow the phase to separate. Store at 4°C in a light-tight bottle

• Iso-propanol or EtOH/Sodium acetate

• 1 mg/ml Glycogen in water

• 10× TE buffer: 100 mM Tris pH 8, 10 mM EDTA pH 8

2.2 Equipment

• Disinfected ⁴ adjustable pipettes, range: 2–20 μl, 20–200 μl, 100–1,000 μl

• Nuclease-free aerosol-resistant pipette tips

• 1.5 ml tubes (autoclaved)

• Single-packed toothpicks

• Sterile or disposable tweezers

• Microtome, with new blade

• Centrifuge suitable for centrifugation of 1.5 ml tubes at 13,200 or 14,000 rpm

• Thermoblock

• Thermomixer (e.g., Eppendorf)

• SpectroPhotometer

2.3 Method

2.3.1 Sample Preparation

• If possible, cool the paraffin blocks at −20°C or on dry ice in aluminium foil in order to cut the sections.

• Using a clean, sharp microtome blade⁵, cut two to ten sections of 5–10 μm thickness depending on the size of the sample. Discard the first section and displace the other ones in 1.5 ml tubes, using a sterile toothpick or tweezers (depending on the section size). Use some sections from a paraffin block without included tissue, treated together with other samples, for negative control analysis.

2.3.2 Deparaffinization (Optional)

• Add 1 ml of xylene,⁶ vortex for 10,” and then maintain the tube at room temperature for approximately 5 min.⁷

• Spin the tube for 5 min at maximum speed (14,000 rpm) in a microcentrifuge and then carefully remove and discard the supernatant using a micropipette or a glass Pasteur pipette.⁸

• Repeat wash with a fresh aliquot of xylene.

• Wash the pellet by adding 1 ml of absolute ethanol. Flick the tubes to dislodge the pellet and then vortex the tubes for 10 s.

• Leave at room temperature for approximately 5 min.

• Spin the tube for 5 min at maximum speed (14,000 rpm) in a microcentrifuge and then carefully remove and discard the supernatant.

• Repeat washes using 90% and 70% ethanol.

• After removing 70% ethanol, allow the tissue pellet to air dry in a thermoblock at 37°C for about 30 min.

2.3.3 Proteolytic Digestion and DNA Extraction

• Add to the tissue pellet 150–300 μl of digestion buffer 1x supplemented with Proteinase K at final concentration of 1 mg/ml. The amount of digestion buffer depends on the tissue amount. The digestion buffer must cover the tissue pellet completely.⁹

• Incubate in the thermomixer for 48–72 h¹⁰ at 55°C, shaking moderately. For longer digestion, Proteinase K can be added again every 24 h.

• Add 1 volume of phenol¹¹-buffered pH 8.0 Tris/CHCl₃ /isoamyl alcohol (50:49:1 v/v/v).¹² Mix well by inverting the tube, and leave on ice for 10–20 min.

• Centrifuge at 14,000 rpm at 4°C for 20 min. The mixture will separate into a lower organic phase, an interphase, and an upper aqueous phase.

• Transfer the upper phase into a new tube, add 1 volume of CHCl₃, mix well for 5 min, and centrifuge at 14,000 rpm at 4°C for 20 min.

• Transfer the supernatant in a new tube containing 5 μl of glycogen solution (1 mg/ml stock) as precipitation carrier. Carefully avoid transferring the interphase containing proteins.

• Precipitate overnight at –20°C with 1 volume of iso-propanol or 2.5 volumes of EtOH supplemented with 0.1 volumes of Sodium acetate 3 M pH 7.

• Centrifuge at 14,000 rpm at 4°C for 20 min and discard the supernatant.

• Wash the pellet with 200 μl of 70% ethanol without resuspending the pellet to wash away the remaining salts.

• Air dry the pellet and resuspend the DNA pellet in the appropriate amount of TE buffer 1×. Store the DNA solution at –20°C.

• For DNA measurement, pipette 199 μl of sterile water into a fresh tube and add 1 μl of DNA extract (dilution factor  =  200). Determine the DNA concentration photometrically at 260 and 280 nm (see Chap. 16, Sect. 16.2.1 for more details).¹³

2.4 Troubleshooting

• If the DNA yield is low, you may have lost the DNA pellet; in such case, repeat the entire process of extraction.

• If the pellet is not visible after centrifugation, the precipitation could have been incomplete because of the absence of a precipitation carrier. Add 5 μl of glycogen 1 mg/ml, and leave at −20°C overnight to complete precipitation.

• If DNA is absent, a nuclease contamination could have occurred. In such case, repeat the extraction using freshly made reagents.