Table 8 Nucleic Acid Extraction Procedures As Described Elsewhere [7, 13]

Common procedures for formalin-fixed, paraffin-embedded (FFPE) samples with histological diagnosis of invasive amebiasis and mycosis

 • Exposure of 25 μm thick sections in 1.5 ml tubes for 2 × 10 min to 1200 μl xylene and for 3 × 10 min to 1200 μl 100% ethanol under gentle constant agitation for deparaffination

 • Discarding of the supernatant following each 10-min step after centrifugation for 10 min at 13,000 g

 • Air-drying of the samples

Specific procedures as applied for the samples with invasive mycosis

 • DNA extraction using the DNA FFPE tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction as follows:

  ○ Proteinase K digestion at 56 °C overnight to get a clear suspension

  ○ Lysis of the fungal cell walls in the pellet with 400 units of Arthrobacter luteus lyticase L2524 (Sigma-Aldrich Corporation, St. Louis, MO, USA) (instead of lyticase L4276 [Sigma-Aldrich Corporation] which was no longer available) per sample for 45 min at 37 °C

Specific procedures as applied for the samples with invasive amebiasis

 • Tissue digestion in 200 μl lysis buffer (0.5% Tween 20 [ICI, American Limited, Merck, Hohenbrunn, Germany], 2 mg/ml proteinase K [Roche, Mannheim, Germany], 3.5 mM MgCl₂, 15 mM ammonium sulfate, and 60 mM Tris/HCl [pH 8.5]) for at least 1 h until a clear lysate was obtained at 56 °C under continuous shaking

 • Boiling for 10 min to stop the digestion by denaturing the proteinase K

 • DNA extraction using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions