DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells

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 µg, 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.

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

“…The statistical analysis of the values regarding the quantity of the gDNA obtained was done with the ''StatsDirect" program. A Kruskal-Wallis test was applied to the gDNA quantity obtained for variance evaluation of independent samples as described by Ribeiro et al (2004).…”

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors

“…Especially during the histopathological preparation, the production of TTIs does not add another laborious procedure besides the snap freezing of tumor tissue, which should be done in any case. The amount of cells on TTIs varied greatly between samples, resulting in a high variation of extracted DNA quantities per slide, as already reported in other studies [6] [7] [8] [10]. Compared to fresh or fresh frozen solid tumor pieces, the cell number on TTIs can be low, which can result in a limited DNA quantity.…”

“…The authors described a number of structural aberrations with a resolution limited by the respective platform [9]. As TTIs can be stored for many years without markedly influencing the quality of extractable DNA [10], these slides are a promising source for molecular studies.…”

Rapid combined light and electron microscopy on large frozen biological samples

“…In general, an EM study can be useful when histochemical analysis raises questions with respect to structural and functional relationships of a cell or a tissue. The present procedure seems to be generally applicable for this purpose because all tissues examined so far (pancreas, liver, colon, and small intestine) showed well-preserved ultrastructure, whereas reproducibility is very high due to its simplicity (Boermeester et al, 1995;Schellens et al, 1995;Van Noorden et al, 1995;Lettinga et al, 1996;Prentø, 1997;Metzger et al, 1998;Ribeiro et al, 2004).…”