PCR amplification of DNA from archival specimens. A methodological approach.
Author(s): Pavelic J, Gall-Troselj K, Bosnar MH, Kardum MM, Pavelic K
Publication: Neoplasma, 1996, Vol. 43, Page 75-81
PubMed ID: 8843966 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 the effects of fixative type on amplification of beta-globin from paraffin-embedded brain specimens.
Summary of Findings:
Amplification of a 318 bp fragment of beta-actin was successful in paraffin-embedded specimens fixed with AmeX, Carnoy's, or 10% buffered formalin. There was less amplification product from specimens fixed with paraformaldehyde or acetone, and PCR was not successful in specimens fixed with Bouin's fixative.
Biospecimens
Preservative Types
- Formalin
- Other Preservative
Diagnoses:
- Neoplastic - Benign
- Neoplastic - Sarcoma
- Normal
- Autopsy
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Paraformaldehyde
Acetone
AMeX
Bouin's fixative
Carnoy's solution
Formalin (buffered)
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Study Purpose
The purpose of this study was to determine the effects of fixation duration, storage, deparaffinization and extraction on the amplification of beta-globin from FFPE brain specimens.
Summary of Findings:
Beta-actin was successfully amplified from unembedded specimens fixed for up to 48 h, but there was a lower product yield when specimens were fixed for 48 h than when fixed for 24 h or less. Amplification of a 720 bp fragment of bcl-2 was successful after 40 cycles in FFPE specimens stored for 1 year, but not in specimens stored for 10 or 39 years. Increasing the number of PCR cycles to 50, allowed for amplification of bcl-2 from 10 year old specimens but not from 39 year old specimens. Beta-actin could be amplified when FFPE specimens were deparaffinized for more than 4 h, but not in specimens deparaffinized for less than 4 h. There was no benefit of extracting the DNA with phenol-chloroform after proteinase k digestion.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Benign
- Neoplastic - Sarcoma
- Normal
- Autopsy
Platform:
Analyte Technology Platform DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 2 h
4 h
6 h
8 h
12 h
24 h
48 h
Biospecimen Preservation Embedding medium Not embedded
Paraffin
Analyte Extraction and Purification Deparaffinization Xylene
1 h
2 h
4 h
24 h
Analyte Extraction and Purification Protein digestion Proteinase k
3 h
4 days
Analyte Extraction and Purification Analyte isolation method Phenol-chloroform extraction
None
PCR Specific Length of gene fragment 318 bp
720 bp
PCR Specific Targeted nucleic acid Beta-actin
Bcl-2
PCR Specific Number of cycles 30 cycles
40 cycles
50 cycles
Storage Storage duration 1 year
10 years
39 years
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Study Purpose
The purpose of this study was to determine the effect of Papanicolaou staining on the amplification of beta-actin and TGF-beta from cytological smears.
Summary of Findings:
Amplification of TGF-beta and beta-actin was only possible from Papanicolaou stained specimens after phenol-chloroform extraction. The authors report that eosin dye migrated through the gel producing an interfering fluorescent signal.
Biospecimens
Preservative Types
- Ethanol
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 Phenol-chloroform extraction
None
PCR Specific Length of gene fragment 318 bp
500 bp
PCR Specific Targeted nucleic acid Beta-actin
TGF-beta
PCR Specific Type of tissue stain Papanicolaou stain