DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells.
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.
Ribeiro
C N M
CN
Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirao Preto, SP, Brazil.
Peres
L C
LC
Pina-Neto
J M
JM
D001344:Autopsy; D002675:Child, Preschool; D000013:Congenital Abnormalities / Q000175:diagnosis*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D022781:Hepatocytes; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D016133:Polymerase Chain Reaction; D013048:Specimen Handling / Q000191:economics / Q000379:methods*; D016707:Tissue Fixation / Q000191:economics / Q000379:methods*
D004247:DNA