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Published on: December 9, 2022
DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells
C N M Ribeiro1, L C Peres, J M Pina-Neto
1Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirao Preto, SP, Brazil.
Abstract:
The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared by each fixation procedure was submitted to DNA extraction with the Wizard genomic DNA purification kit for DNA quantification and five of the preparations were amplified by multiplex PCR (azoospermia factor genes). The amount of DNA extracted varied from 20 to 8,640 microg, with significant differences between fixation methods. Scrape preparation fixed in 95% ethanol provided larger amount of extracted DNA. However, the mean for all groups was higher than the quantity needed for PCR (50 ng) or Southern blot (500 ng). There were no qualitative differences among the different material and fixatives. The same results were also obtained for glass slides stored at room temperature for 6, 12, 18 and 24 months. We conclude that touch and scrape preparations fixed in 95% ethanol are a good source of DNA and present fewer limitations than cell culture, tissue paraffin embedding or freezing that require sterile material, culture medium, laboratory equipment and trained technicians. In addition, they are more practical and less labor intensive and can be obtained and stored for a long time at low cost.
Insights
This study presents a simple, low-cost method for collecting and fixing pediatric autopsy cells, yielding sufficient DNA for genetic analysis. 95% ethanol fixation of scrape preparations proved most effective for DNA extraction.
Area of Science:
- Forensic Science
- Molecular Biology
- Genetics
Background:
- Accurate DNA extraction from pediatric autopsy samples is crucial for genetic studies.
- Traditional methods for sample preservation can be costly and labor-intensive.
Purpose of the Study:
- To develop a simplified, cost-effective method for collecting and fixing pediatric autopsy cells.
- To assess the quantitative and qualitative adequacy of DNA extracted using this method.
Main Methods:
- Touch and scrape preparations of pediatric liver cells from 15 autopsies were fixed using 95% ethanol or methanol:acetic acid.
- DNA was extracted using the Wizard genomic DNA purification kit and quantified.
- Multiplex PCR was used to amplify azoospermia factor genes from a subset of samples.
Main Results:
- DNA yield ranged from 20 to 8,640 µg, with 95% ethanol fixation of scrape preparations yielding the most DNA.
- Extracted DNA quantities exceeded requirements for PCR (50 ng) and Southern blot (500 ng).
- No qualitative DNA differences were observed between fixatives or storage durations up to 24 months.
Conclusions:
- Touch and scrape preparations fixed in 95% ethanol provide a practical, low-cost, and reliable source of DNA from pediatric autopsy samples.
- This method offers advantages over cell culture, paraffin embedding, or freezing, requiring less specialized equipment and expertise.
- The method is suitable for long-term storage and genetic analyses, including PCR and Southern blot.
