The Role of DNase and EDTA on DNA Degradation in Formaldehyde Fixed Tissues

Abstract

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.

Whole genome amplification for CGH analysis: Linker‐adapter PCR as the method of choice for difficult and limited samples

“…Our data, rather, suggest proper handling of the surgery specimen as a key requisite for DNA-amplification and CGH analysis. It is of utmost importance that liver samples are fixed immediately after surgery and that the fixation time is long enough, depending on the sample size (20). This is additionally corroborated by the fact that we monitored distinct differences in success rates between samples obtained from different pathology departments.…”

Calmodulin priming: Nuclear translocation of a calmodulin complex and the memory of prior neuronal activity

“…1). Previous reports have demonstrated that aldehyde fixation causes an immediate and massive cytosolic Ca 2ϩ increase (35), thus allowing fast Ca 2ϩ -dependent processes to proceed during fixation (36). We buffered external Ca 2ϩ in the fixative with 4 mM EGTA to prevent the paraformaldehyde solution from acting as a trigger for CaM translocation.…”

Obtaining, Storing and Archiving Specimens and Tissue Samples for Use in Molecular Studies

“…The average length of the DNA from ancient soft tissue is less than 200 base-pairs (bp) [22]. This reduction in length is partially attributable to strand breakage caused by autolytic processes (e.g., DNAse activity) that occur rapidly after death [19,23,24]. However, equally important sources of damage are subsequent oxidative and hydrolytic effects that either break, or labilise, phosphodiester and carbon-nitrogen (sugar-base) bonds [21,25,26].…”

“…For example, Greer et al [64] found that fragments of DNA, approximately 1 kb in length, were impossible to amplify from formalin-fixed tissues after only 24 hours at ambient temperature. However, high molecular weight DNA was obtained from tissues fixed at 4 °C in formalin buffered with 4 M urea [23,129]. Similarly, Noguchi et al [63] established that fixation in formalin at 4 °C and in formalin containing 5 mmol/L EDTA at ambient temperature preserved significantly more high molecular weight DNA than fixation in formalin at ambient temperature.…”