Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues.

Author(s): An SF, Fleming KA

Publication: J Clin Pathol, 1991, Vol. 44, Page 924-7

PubMed ID: 1752983 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare PCR amplification of DNA purified by different methods from FFPE tissues. Tissue specimens used were liver, skin, rectum, ovary, bladder, omentum, cervix, uterus, rectal polyp, appendix, stomach, thyroid, heart, prostate, gum, lymph node and kidney.

   Summary of Findings:

   It was possible to amplify the beta-globin gene directly from frozen tissue specimens but not FFPE specimens. Boiling and digesting with proteinase K were not sufficient to allow for amplification from FFPE specimens, but when combined with a phenol/chloroform extraction and filtration through a centricon membrane amplification was possible when between 50 ng and 2 ug of target DNA was used. The authors report that a nested PCR was necessary for amplification of a 227 bp fragment, but that a 110 bp fragment did not require a second round of amplification.

   Biospecimens

   • Tissue - Skin
   • Tissue - Liver
   • Tissue - Colorectal
   • Tissue - Ovary
   • Tissue - Bladder
   • Tissue - Cervix
   • Tissue - Uterus
   • Tissue - Appendix
   • Tissue - Stomach
   • Tissue - Thyroid Gland
   • Tissue - Heart
   • Tissue - Prostate
   • Tissue - Oral Cavity
   • Tissue - Lymph Node
   • Tissue - Kidney
   • Tissue - Other

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte purification	No additional procedure after dewaxing Boiled for 20 minutes Boiled 20 min, passed through sephadex G50 column Boiled for 20 min digested with proteinase K Boiled for 20 min, proteinase K, phenol-chloroform extract Boiled for 20 min, proteinase K, phenol-chloroform extract, centricon filtration
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh) Frozen
   PCR Specific	Targeted nucleic acid	Human beta globin
   PCR Specific	Length of gene fragment	355/227 bp 110 bp

   View more