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.
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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)