Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Residual Humidity in Paraffin Embedded Tissue Reduces Nucleic Acid Stability

Peter M. Abuja
* ORCID logo ,
Daniela Pabst
,
Benjamin Bourgeois
,
Martina Loibner
,
Christine Ulz
,
Iris Kufferath
,
Ulrike Fackelmann
,
Conny Stumptner
ORCID logo ,
Rainer Kraemer
,
Tobias Madl
* ORCID logo ,
Kurt Zatloukal
ORCID logo
Version 1 : Received: 22 December 2022 / Approved: 23 December 2022 / Online: 23 December 2022 (09:04:08 CET)

A peer-reviewed article of this Preprint also exists.

Abuja, P.M.; Pabst, D.; Bourgeois, B.; Loibner, M.; Ulz, C.; Kufferath, I.; Fackelmann, U.; Stumptner, C.; Kraemer, R.; Madl, T.; et al. Residual Humidity in Paraffin-Embedded Tissue Reduces Nucleic Acid Stability. International Journal of Molecular Sciences 2023, 24, 8010, doi:10.3390/ijms24098010. Abuja, P.M.; Pabst, D.; Bourgeois, B.; Loibner, M.; Ulz, C.; Kufferath, I.; Fackelmann, U.; Stumptner, C.; Kraemer, R.; Madl, T.; et al. Residual Humidity in Paraffin-Embedded Tissue Reduces Nucleic Acid Stability. International Journal of Molecular Sciences 2023, 24, 8010, doi:10.3390/ijms24098010.

Abstract

Molecular diagnostics in healthcare relies increasingly on genomic and transcriptomic method-ologies and requires appropriate tissue specimens from which nucleic acids (NA) of sufficiently high quality can be obtained. Besides suration of ischemia and fixation type, NA quality depends on a variety of other pre-analytical parameters, like storage conditions and duration. It has been discussed that improper dehydration of tissue during processing influences quality of NAs and shelf-life of fixed tissue. Here we report on establishing a method to determine the amount of residual water in fixed, paraffin-embedded tissue (fixed by neutral buffered formalin, or a non-crosslinking fixative) and its correlation to the performance of NAs in qRT-PCR analyses. The amount of residual water depended primarily on the fixative type and the dehydration pro-tocol and, to a lesser extent on storage conditions and time. Moreover, we found that these pa-rameters were associated with the qRT-PCR performance of extracted NAs. Besides cross-linking of NAs and modification of nucleobases by formalin, hydrolysis of NAs by residual water was found to contribute to reduced qRT-PCR performance. The negative effects of residual water on NA stability are not only important for the design and interpretation of research, but must also be taken into account in clinical diagnostics where reanalysis of archived tissue from a primary tumour may be required, e.g., after disease recurrence. We conclude that improving shelf-life of fixed tissue requires meticulous dehydration and dry storage to minimize the degradative in-fluence of residual water on NAs.

Keywords

fixed tissue; nucleic acid quality; next-generation sequencing,

Subject

Medicine and Pharmacology, Pathology and Pathobiology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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