Abstract
The aim of the present study was to examine an ultrasound-accelerated fixation technique that reduces the exposure time of the tissue to formaldehyde with respect to the analysis of nucleic acids. We extracted and analysed DNA and RNA from three series of autopsy specimens from five routine cases. Two series were shortly fixed in 4% buffered formalin (15 and 30 min, respectively) whilst being irradiated with high-frequency, high-intensity ultrasound. The last series (control) was routinely fixed in 4% buffered formalin for 24–48 h without irradiation. Although sufficient amounts of DNA of good quality could be extracted and amplified from all three series, the peak heights obtained from conventional fixation were smaller and allele dropout occurred more often, especially for the longer amplicons. RNA yield depended on the fixation procedure, i.e. the shortest fixation time led to the highest RNA yield and quality. No differences were observed with regard to the quality of the histological slides both with conventional and immunohistochemical staining methods. Keeping in mind the increasing need for molecular diagnosis, this fixation technique can be useful to ensure stable quality of nucleic acids in archived autopsy specimens.
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Acknowledgements
The authors would like to thank Beate Annuß, Kirsten Waterkamp and Ulla Sibbing of the Institute in Münster for their excellent technical support. Thanks to Ulrike Schmidt and Sabine Lutz-Bonengel of the Institute in Freiburg for their helpful comments on the manuscript.
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Tony Fracasso and Marielle Heinrich contributed equally to this work.
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Table S1
Comparison of the normalised peak height at different loci by Student’s t test (http://www.physics.csbsju.edu/stats/t-test_bulk_form.html). Only the significant results are shown (DOC 26 kb)
Table S2a
DNA concentration for each sample and peak height at each amplified locus (XLS 62 kb)
Table S2b
Comparison of the normalised peak heights for each group at each locus by Student’s t test. The significant results are depicted in green, the non-significant in red (XLS 50 kb)
Table S2c
Allelic profile of the investigated cases (XLS 39 kb)
Fig. S1
a Section of the lung fixed for 15 min. Actin, ×100. b Section of the lung conventionally fixed. Actin, ×100 (PPT 3.13 mb)
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Fracasso, T., Heinrich, M., Hohoff, C. et al. Ultrasound-accelerated formalin fixation improves the preservation of nucleic acids extraction in histological sections. Int J Legal Med 123, 521–525 (2009). https://doi.org/10.1007/s00414-009-0368-1
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DOI: https://doi.org/10.1007/s00414-009-0368-1