Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction.
Author(s): Rogers BB, Alpert LC, Hine EA, Buffone GJ
Publication: Am J Pathol, 1990, Vol. 136, Page 541-8
PubMed ID: 2156429 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine if pretreatment of formalin-fixed, paraffin-embedded (FFPE) biopsy gastrointestinal specimens or the size of liver autopsy specimens impact the efficiency of PCR amplification of the c-K-ras gene.
Summary of Findings:
Tissue permeabilization with proteinase K or Triton X-100 (overnight at 48 degrees C) prior to DNA extraction did not significantly affect amplification of c-K-ras when compared to negative controls. While similar amplicon levels were observed among differing specimen sizes (assessed by proportions of a 5 um tissue section) fixed in formalin for 24 h, the generation of nonspecific products decreased with decreasing specimen size.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Not specified
- Autopsy
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 24 h
Analyte Extraction and Purification Cell/tissue permeabilization No pretreatment
Proteinase K
Triton X-100
Biospecimen Aliquots and Components Aliquot size/volume 1/6 of a tissue section
Entire tissue section
1/2 of a tissue section
1/4 of a tissue section
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Study Purpose
The purpose of this study was to determine if the duration of formalin fixation (6 h to 4 weeks) affects PCR amplification efficiency of c-K-ras.
Summary of Findings:
PCR amplification efficiency decreased in a fixation duration dependent manner. While the earliest decrease in amplicon intensity was observed in specimens fixed for 72 hours, a more robust and significant decrease was observed after fixation in formalin for 1 week or more (4 weeks). Paraffin embedding had no observable effect on PCR amplification.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Autopsy
- Not specified
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 6 h
24 h
72 h
1 week
2 weeks
3 weeks
4 weeks
Biospecimen Preservation Embedding medium OCT
Paraffin
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Study Purpose
The purpose of this study was to determine if the method of biospecimen acquisition (biopsy versus autopsy) affects PCR amplification success of DNA extracted from FFPE tissue specimens. Liver, lung, heart, colon, and gastroitestinal tract specimens were included in the study.
Summary of Findings:
Of the 6 autopsy specimens assessed 2 failed to generate a c-K-ras amplicon, while all 39 biopsy specimens resulted in a detectable product. The duration of formalin fixation of these specimens was not specified.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Autopsy
- Not specified
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA PCR DNA Southern blot Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Method of tissue acquisition Autopsy
Biopsy