Residual Humidity in Paraffin-Embedded Tissue Reduces Nucleic Acid Stability

Abstract

Molecular diagnostics in healthcare relies increasingly on genomic and transcriptomic methodologies and requires appropriate tissue specimens from which nucleic acids (NA) of sufficiently high quality can be obtained. Besides the duration of ischemia and fixation type, NA quality depends on a variety of other pre-analytical parameters, such as storage conditions and duration. It has been discussed that the improper dehydration of tissue during processing influences the quality of NAs and the shelf life of fixed tissue. Here, we report on establishing a method for determining 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 quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The amount of residual water depended primarily on the fixative type and the dehydration protocol and, to a lesser extent, on storage conditions and time. Moreover, we found that these parameters were associated with the qRT-PCR performance of extracted NAs. Besides the cross-linking of NAs and the modification of nucleobases by formalin, the 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 the reanalysis of archived tissue from a primary tumor may be required (e.g., after disease recurrence). We conclude that improving the shelf life of fixed tissue requires meticulous dehydration and dry storage to minimize the degradative influence of residual water on NAs.