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

C.N.M Ribeiro,L.C Peres,J.M Pina-Neto

doi:10.1590/s0100-879x2004000500002

C.N.M Ribeiro, L.C Peres + Show 1 more

Open Access

https://doi.org/10.1590/s0100-879x2004000500002

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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.

Highlights

Congenital anomalies are structural, functional, biochemical or behavioral alterations observable at birth
The touch and scrape preparatives used in the present study were obtained from 15 children of both sexes ranging in age from 25 min to four years who presented some type of congenital anomaly, and submitted to autopsy at the Pathology Service of the University Hospital, Faculty of Medicine of Ribeirão Preto (HCFMRP-USP), from July to December 2001
Pair-wise comparison of the samples (Table 3) showed that there was a significant difference between TP95 and SP95, TPMA and SP95 and SP95 and SPMA, indicating that 95% ethanol is a better fixative than methanol:acetic acid and that scrape preparatives are better than touch preparatives

Summary

Introduction

Congenital anomalies are structural, functional, biochemical or behavioral alterations observable at birth. They involve about 3% of liveborns in the general population [1] and represent the major cause of post-neonatal death in the city of Ribeirão Preto [2], corresponding to about 25% of the cases of pediatric autopsy at the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo [3]. Many of the anomalies observed at autopsy can be diagnosed by dysmorphologic examination to establish the syndromic and etiological diagnosis, other procedures are often necessary, ranging from cytogenetics to biochemical and/or molecular genetic techniques. Non-numerical chromosome alterations or alterations below the level of optical resolution (microdeletions) cannot be identified by this method but require molecular techniques

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