DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections

Author: Shi et al.

Source: Contributed by APostodoc
Abstract: Describes two methods of extracting DNA from archived, paraffin-embedded sections. One is the standard non-heating enzymatic digestion method; the other is heating method based on the principles of the antigen retrieval technique, which yields better DNA quantity.
Date Added: 2004-05-06
Date Modified: 2004-05-06

 

Section 1. Non-heating DNA Extraction Protocol

  1. Cut paraffin block at 10 µm and collected in an autoclaved plastic microtube (1.5 ml).
  2. Add 1 ml xylene to the microtube containing tissue sections for 30 min for two changes,
  3. Add 100% and 75% ethanol for 30 min with two changes
  4. Wash with PBS for 15 min with two changes
  5. Add 500 µl of lysis buffer (proteinase K 20 mg/ml, 50 µl, 1 M Tris-HCl solution 10 µl, 0.5 M EDTA 2 µl, 10% SDS 100 µl, and distilled water 838 ml)
  6. Incubated at 52°C overnight until all tissue fragments were dissolved completely
  7. Add 500 µl phenol:chloroform:isopropanol alcohol at 25:24:1 to the de-waxed tissue
  8. Mix by vortex
  9. Centrifugation at RT, 12,000 x g for 10 min
  10. Transfer the supernatant fluid to an autoclaved microtube using a 100-µl pipette
  11. Add one volume of chloroform to the supernatant, mixed by vortexing
  12. Centrifuged at 12,000 x g for 5 min.
  13. Carefully remove the upper aqueous supernatant to another fresh microtube
  14. Adding 0.1 volume of 3 M sodium acetate to the new tube
  15. Mix by vortexing
  16. Add 1 volume of isopropanol, and incubate at -20C overnight.
  17. The precipitated DNA was centrifuged at 12,000 x g at 4C.
  18. Discard the supernatant fluid and wash once with 75% ethanol.
  19. Collect the extracted DNA after further centrifugation.
  20. Dissolve the final yield of DNA in 50 µl distilled water after drying completely in a hood.

 

Section 2. Heating Protocol for DNA Extraction

 

Overview

According to Shi et al, heating before following the standard protocol above can get high DNA yield.

Materials and Reagents

  1. Universal Buffer Solution

Add  28.6 mM of each chemical

Adjust pH to 9 using 0.2 N sodium hydroxide

Procedure

  1. Add 500 µl universal buffer solution at pH 9 to a microtube containing a 20-µm tissue section
  2. Heat at 120°C using an autoclave for 20 min
  3. Allow the tube to cool for 5 min
  4. Follow the standard protocol above but omitting the enzyme digestion step

 

Reference

Shi et al, Journal of Histochemistry and Cytochemistry, Vol. 50, 1005-1011, August 2002.

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