Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity

PLOS ONE, Jun 2011

Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues.

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Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity

Sapino A (2011) Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity. PLoS ONE 6(6): e21043. doi:10.1371/journal.pone.0021043 Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity Gianni Bussolati 0 Laura Annaratone 0 Enzo Medico 0 Giuseppe D'Armento 0 Anna Sapino 0 Chun-Ming Wong, University of Hong Kong, Hong Kong 0 1 Department of Biomedical Sciences and Human Oncology, University of Turin , Turin , Italy , 2 Department of Oncological Sciences, Institute for Cancer Research and Treatment (IRCC), University of Turin , Candiolo , Italy Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4uC until fixation and then fixed at 4uC, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues. - Since its introduction as a histological fixative back in the 19th century [1], the 4% formaldehyde solution in water (formalin) has been adopted as the fixative of choice in histopathology. However, the uses of formalin-fixed tissues have varied over time. Originally, optimal morphological preservation was the sole requirement, but in more recent times, with the advent of immuno-histochemical typing, reliable antigenic preservation is also required [2,3]. As a consequence, the protocols of formalin fixation have become stricter. This issue is particularly relevant in onco-pathology for the evaluation of factors predicting responsiveness to therapeutic treatments, and thus, fixation in phosphate buffered formalin (PBF) of breast cancer tissue blocks for no less than 6 and no more than 48 hours is now required in order to guarantee an optimal evaluation of Estrogen (ER) and Progesterone Receptors (PgR) and HER2 expression [4,5]. In more recent times, a crucial request in cancer pathology has been nucleic acid preservation from formalin-fixed paraffinembedded (FFPE) tissues because large tissue archives would thus be available for gene expression profiling, with the goal of generating new and reliable diagnostic and prognostic parameters [6,7]. The first and most crucial issue that must be addressed for molecular gene signatures is ensuring that the sample is properly collected to obtain good quality RNA. The accuracy of molecular tests is utterly dependent on careful preservation of biological samples prior to analysis and then on an adequate sampling of fresh tissues that have to be representative of and enriched for the tumor cell population in order to obtain the specific RNA target. Studies conducted on the preservation status of nucleic acids in FFPE tissues generally agree on the relatively good (though not optimal) preservation of DNA [8]. On the contrary, RNA has been found to be heavily degraded and fragmented so that only short sequences (approximately 100200 nucleotides) can be recognized and amplified [913]. The reasons for this effect are presently unknown, but some hints can be derived from the numerous studies conducted on the reaction of formaldehyde with different tissue components and specifically with nucleic acids [14 16].The main effect of formaldehyde in tissues is linked to the formation of methylol groups on amino groups first, followed by the establishment of cross-linking methylene groups that lead to proper fixation [13]. Bases of nucleic acids are involved in this process, resulting in cross-linking with side-chain amino groups of proteins. However, this linkage is at least partly reversible following extensive treatment of FFPE tissue sections with peptidases and high temperature [9,13,17,18]. We conclude that cross-linking of nucleic acid bases cannot be the sole responsible for nucleic acid fragmentation and degradation. Chung YJ et al. [9] have demonstrated that substantial RNA degradation may occur during the so called warm ischemia that refers to the time of transfer from an operation room (or removal of blood supply) to pathology laboratory. The RNA degradation due to warm ischemia may be slowed down by cooling the specimen. The ischemic process may continue during fixation [9]. In fact, while formalin penetration is a rather fast process, t (...truncated)


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Gianni Bussolati, Laura Annaratone, Enzo Medico, Giuseppe D'Armento, Anna Sapino. Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity, PLOS ONE, 2011, 6, DOI: 10.1371/journal.pone.0021043