Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.
Author(s): Jackson DP, Lewis FA, Taylor GR, Boylston AW, Quirke P
Publication: J Clin Pathol, 1990, Vol. 43, Page 499-504
PubMed ID: 1696290 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to comparatively assess different DNA extraction methods for DNA yield and quality in both fresh frozen and formaldehyde-fixed, paraffin-embedded specimens. Tonsil and spleen were analyzed.
Summary of Findings:
For frozen specimens, a 1 h proteinase K incubation yielded DNA of equivalent quality and quantity as longer incubations and yielded superior DNA for use in PCR than DNA extraction with SDS or boiling. For formaldehyde-fixed, paraffin-embedded specimens, a five day incubation with proteinase K generated the greatest yield of high molecular weight DNA compared with other methods and incubation times. Deparaffinization with xylene did not affect DNA yield or quality. Compared to specimens fixed for 24 h in formaldehyde, fresh frozen specimens yielded 40 times more DNA of a higher molecular weight.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Fluorometry DNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Proteinase K (0.1 mg/ml)
SDS incubation
Boiling
Analyte Extraction and Purification Deparaffinization Xylene
Untreated
Biospecimen Preservation Type of fixation/preservation Formaldehyde
Snap frozen
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Study Purpose
The purpose of this study was to determine if successful amplification of the factor VIII gene by PCR is influenced by the extraction method used for specimens fixed in 4% formaldehyde and paraffin embedded. Tonsil and spleen specimens were assessed.
Summary of Findings:
PCR amplification of a 142 bp fragment of the factor VIII gene was successful for all extraction methods using specimens fixed in 4% formaldehyde and paraffin embedded, although the intensity of the band was dependent on the concentration of the template.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Proteinase K (0.1 mg/ml)
SDS incubation
Boiling
-
Study Purpose
The purpose of this study was to compare the effects of different fixation conditions (fixative type, temperature, duration) and a fixation delay at room temperature on the quantity, quality, and successful PCR amplification of extracted DNA. Tonsil and spleen were analyzed.
Summary of Findings:
The greatest yields of high molecular weight DNA were obtained for specimens that had been fixed in formaldehyde or Carnoy's reagent. PCR analysis was successful for 4 of the 6 fixatives examined: 4% formaldehyde, neutral buffered formalin, Bouin's reagent, and Carnoy's reagent. A fixation delay of 7 days at room temperature resulted in a modest degree of DNA degradation. Extended fixation (12 weeks) adversely affected DNA quality resulting in more extensive degradation. The temperature of fixation greatly influenced DNA quality, with increased degradation observed at fixation temperatures of 37 and 60 degrees C.
Biospecimens
Preservative Types
- Formalin
- Other Preservative
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Fluorometry DNA Electrophoresis DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Bouin's fixative
Carnoy's solution
Formaldehyde
Formalin (buffered)
Formol sublimate
Paraformaldehyde
Biospecimen Preservation Time in fixative 24 h
12 weeks
Biospecimen Preservation Temperature of fixation/preservation Room temperature
37 degrees C
60 degrees C
Storage Time at room temperature 0 d
7 d