DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: Heating under the influence of pH

Shan Rong Shi, Richard J Cote, Lin Wu, Cheng Liu, Ram Datar, Yan Shi, Dongxin Liu, Hyoeun Lim, Clive R. Taylor

Research output: Contribution to journalArticle

195 Citations (Scopus)

Abstract

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.

Original languageEnglish
Pages (from-to)1005-1011
Number of pages7
JournalJournal of Histochemistry and Cytochemistry
Volume50
Issue number8
StatePublished - Aug 10 2002
Externally publishedYes

Fingerprint

Paraffin
Heating
Formaldehyde
Antigens
DNA
Polymerase Chain Reaction
Photometry
Surgical Pathology
Palatine Tonsil
Nucleic Acids
In Situ Hybridization
Colon
Proteins
Hot Temperature
Lymph Nodes
Staining and Labeling
Temperature
Acids

Keywords

  • Antigen retrieval
  • DNA extraction
  • Formalin-fixed, paraffin-embedded tissue
  • PCR

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle : Heating under the influence of pH. / Shi, Shan Rong; Cote, Richard J; Wu, Lin; Liu, Cheng; Datar, Ram; Shi, Yan; Liu, Dongxin; Lim, Hyoeun; Taylor, Clive R.

In: Journal of Histochemistry and Cytochemistry, Vol. 50, No. 8, 10.08.2002, p. 1005-1011.

Research output: Contribution to journalArticle

Shi, Shan Rong ; Cote, Richard J ; Wu, Lin ; Liu, Cheng ; Datar, Ram ; Shi, Yan ; Liu, Dongxin ; Lim, Hyoeun ; Taylor, Clive R. / DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle : Heating under the influence of pH. In: Journal of Histochemistry and Cytochemistry. 2002 ; Vol. 50, No. 8. pp. 1005-1011.
@article{474492af5c174f7da575af54e024e127,
title = "DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: Heating under the influence of pH",
abstract = "During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.",
keywords = "Antigen retrieval, DNA extraction, Formalin-fixed, paraffin-embedded tissue, PCR",
author = "Shi, {Shan Rong} and Cote, {Richard J} and Lin Wu and Cheng Liu and Ram Datar and Yan Shi and Dongxin Liu and Hyoeun Lim and Taylor, {Clive R.}",
year = "2002",
month = "8",
day = "10",
language = "English",
volume = "50",
pages = "1005--1011",
journal = "Journal of Histochemistry and Cytochemistry",
issn = "0022-1554",
publisher = "Histochemical Society Inc.",
number = "8",

}

TY - JOUR

T1 - DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle

T2 - Heating under the influence of pH

AU - Shi, Shan Rong

AU - Cote, Richard J

AU - Wu, Lin

AU - Liu, Cheng

AU - Datar, Ram

AU - Shi, Yan

AU - Liu, Dongxin

AU - Lim, Hyoeun

AU - Taylor, Clive R.

PY - 2002/8/10

Y1 - 2002/8/10

N2 - During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.

AB - During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.

KW - Antigen retrieval

KW - DNA extraction

KW - Formalin-fixed, paraffin-embedded tissue

KW - PCR

UR - http://www.scopus.com/inward/record.url?scp=0036321629&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036321629&partnerID=8YFLogxK

M3 - Article

C2 - 12133903

AN - SCOPUS:0036321629

VL - 50

SP - 1005

EP - 1011

JO - Journal of Histochemistry and Cytochemistry

JF - Journal of Histochemistry and Cytochemistry

SN - 0022-1554

IS - 8

ER -