Extraction of DNA from paraffin blocks for Southern blot analysis.
Author(s): Mies C, Houldsworth J, Chaganti RS
Publication: Am J Surg Pathol, 1991, Vol. 15, Page 169-74
PubMed ID: 1989465 PubMed Review Paper? No
Purpose of Paper
This paper compared Southern blot hybridization of DNA extracted from formalin-fixed and paraffin-embedded (FFPE) and frozen specimens.
Conclusion of Paper
FFPE specimens were of sufficient quality and integrity for identification of immunoglobulin heavy chain rearrangements in lymphoma specimens and detection of human epidermal growth factor receptor 2 (HER-2) copy number changes in gastric carcinoma but the intensity of the bands was generally lower than for matched frozen specimens and the authors reported slight changes in the apparent size of some restriction endonuclease fragments.
Studies
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Study Purpose
This study compared Southern blot hybridization of DNA extracted from paired FFPE and frozen specimens from 11 normal tissues (including liver, breast, testis, myometrium, and ovary), 18 lymphoid lesions, and 9 gastric carcinoma tumors (from five patients). Fresh 50-100µm sections of FFPE specimens were deparaffinized in Histoclear and proteinase K digested for 48-72 h at 45˚C. Frozen specimens were minced and proteinase K digested for 18-24 h at 45˚C. DNA was extracted from lysates using phenol/chloroform with some specimens. DNA was digested with HinDIII (for immunoglobulin heavy chain) or EcoRI (for HER-2/neu), electrophoresed, and transferred by vacuum or capillary to nylon membranes.
Summary of Findings:
Although FFPE specimens were of sufficient quality and integrity for successful Southern blot hybridization of the immunoglobulin heavy chain (11 kb unrearranged) and HER-2/Neu (12.5 kb) in most specimens, the intensity of the bands was generally lower than for the matched frozen specimens, particularly in the gastric carcinoma specimens. The authors also reported slight changes in the apparent size of some restriction endonuclease fragments in the FFPE specimens compared to the matched frozen specimens. Southern blot analysis of FFPE specimens allowed for identification of the immunoglobulin heavy chain rearrangements in lymphoma specimens and for detecting HER-2 copy number changes in gastric carcinoma specimens.
Biospecimens
- Tissue - Breast
- Tissue - Stomach
- Tissue - Liver
- Tissue - Ovary
- Tissue - Lymph Node
- Tissue - Uterus
- Tissue - Testis
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Lymphoma
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Southern blot Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Southern blot Specific Targeted nucleic acid Immunoglobulin heavy chain locus
T-cell receptor β (TCR-β)
Her-2/neu
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen