Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

help |

DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat induced retrieval in an alkaline solution

Author(s): Shi Shan-Rong, Datar Ram, Liu Cheng, Wu Lin, Zhang Zina, Cote Richard J, Taylor Clive R

Publication: HistochemCell Biol, 2004, Vol. 122, Page 211

PubMed ID: 15322858 PubMed Review Paper? No

Purpose of Paper

To assess the efficiency of several antigen retrieval solutions (NaOH, KOH, Britton and Robinson buffer, SDS, Tween 20, guanidine thiocyanate) in various concentrations and mixtures, in the quality, yield, and molecular applicability of the DNA extracted from formalin fixed paraffin embedded (FFPE) tissues.

Conclusion of Paper

Antigen retrieval of FFPE tissue using high temperature solutions (100-120 degrees C) containing 0.1M NaOH and 0.1% guanidine thiocyanate (GITC) or 1% SDS were most efficient in the extraction of large amounts of high quality DNA. Storage of extracted DNA for 1 month at 4 degrees C did not affect molecular analyses.

Studies

Study Purpose

A comparative analysis of the yield of DNA extracted from FFPE tissues (lymph node, colon, small intestine, breast, and bladder as well as benign and cancerous) with differing (1) antigen retrieval solutions, (2) concentrations of solution components, and (3) solution temperatures.

Summary of Findings:

Antigen retrieval solutions resulting in the greatest DNA yield were 0.1% GITC with 0.1M NaOH, 0.1M NaOH, and 1% SDS with 0.1M NaOH. Generally, higher antigen retrieval incubation temperatures (120 degrees C) increased DNA yield.

Biospecimens

Preservative Types

Formalin

Diagnoses:

Neoplastic - Benign
Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
DNA	Spectrophotometry

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Antigen retrieval	NaOH KOH Britton and Robinson Buffer SDS Tween 20 GITC 0.01M Solution concentration 0.1 M Solution concentration 1% Solution concentration 0.5% Solution concentration 10% Solution concentration
Analyte Extraction and Purification	Temperature of heat-induced retrieval	80 degrees C 100 degrees C 120 degrees C

Study Purpose

A comparative analysis of the integrity of DNA extracted from FFPE tissues (lymph node, colon, small intestine, breast, and bladder as well as benign and cancerous) with differing (1) antigen retrieval solutions, (2) concentrations of solution components, and (3) solution temperatures.

Summary of Findings:

DNA integrity, as determined by visual examination of electrophoretic patterns, was better preserved after antigen retrieval with 0.1 M NaOH, 0.1 M KOH, and 0.1% GITC with 0.1 M NaOH.

Biospecimens

Preservative Types

Formalin

Diagnoses:

Neoplastic - Benign
Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
DNA	Electrophoresis

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Antigen retrieval	NaOH KOH 0.01 M Solution concentration 0.1 M Solution concentration 1% Solution concentration 10% Solution concentration 0.5% Solution concentration Britton and Robinson buffer SDS Tween 20 GITC

Study Purpose

The applicability of DNA extracted from FFPE tissue with differing (1) antigen retrieval solutions, (2) concentrations of solution components, and (3) solution temperatures for molecular analyses was examined using semi-quantitative PCR and real time kinetic thermocycling (KTC) PCR.

Summary of Findings:

Semi-quantitative PCR results were variable among gene targets and antigen retrieval solutions; however, in general 0.1 M NaOH or 0.1 M KOH alone or in combination with other chemicals yielded strong, discrete, and appropriate bands. Further, antigen retrieval at higher temperatures (100-120 degrees C) increased band intensity and PCR efficiency, particularly when generating longer amplicons. Results of KTC PCR analysis correlated well with semi-quantitative PCR results; 0.1% GITC with 0.1 M NaOH, 1% SDS with 0.1 M NaOH, and 0.5% Tween 20 with 0.1 M NaOH yielded the greatest quantity of PCR products. Storage of extracted DNA at 4 degrees C for 1 month did not impact PCR results.

Biospecimens

Tissue - Lymph Node

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma
Neoplastic - Benign

Platform:

Analyte	Technology Platform
DNA	PCR
DNA	Real-time qPCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Antigen retrieval	NaOH KOH 0.01 M Solution concentration 0.1 M Solution concentration 1% Solution concentration 10% Solution concentration 0.5% Solution concentration Britton and Robinson buffer SDS Tween 20 GITC
Analyte Extraction and Purification	Temperature of heat-induced retrieval	80 degrees C 100 degrees C 120 degrees C

You Recently Viewed

News and Announcements

Most Downloaded SOPs in 2024

New Articles on the GTEx Project are Now FREELY Available!

Just Published!

More...