Degradation of Macromolecules during Preservation of Lyophilized Pathological Tissues

Takahashi, Rei; Matsuo, Shingo; Okuyama, T.; Sugiyama, Taketoshi

doi:10.1016/s0344-0338(11)80729-6

Cited by 12 publications

(12 citation statements)

References 16 publications

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“…22 Using Western blot analyses, we could not detect any protein degradation of lyophilized liver tissue after 6 months of storage. In this study we demonstrated the feasibility of using lyophilized cytologic specimens for immunostaining.…”

Section: Discussionmentioning

confidence: 80%

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Freeze Substitution and Freeze Drying for Stable, Long-Term Preservation of Cytologic Specimens for Immunostaining

Takahashi¹,

Okuyama²,

Matsuo³

et al. 1996

Acta Cytologica

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Section: Discussionmentioning

confidence: 80%

“…22 The method basically is a modification of standard lyophilization, which is used for preservation of freeze-dried, resin-embedded tissues. 10,11 Our method of drying cytologic specimens allows them to be stored for months at room temperature.…”

mentioning

confidence: 99%

Freeze Substitution and Freeze Drying for Stable, Long-Term Preservation of Cytologic Specimens for Immunostaining

Takahashi¹,

Okuyama²,

Matsuo³

et al. 1996

Acta Cytologica

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“…We have investigated the feasibility of using shelf‐stable preservation; that is, storing freeze‐dried tissues at room temperature, as an alternative method of preserving tissue samples for various molecular analyses at the DNA, RNA, and protein levels. Previous studies have found no visible degradation of DNA by agarose gel electrophoresis after 24 weeks of storage, but mild degradation occurred after 1 year of storage 13 , 14 . The degradation of total RNA was more severe, however, and occurred more quickly.…”

mentioning

confidence: 74%

Preservation of pathological tissue specimens by freeze‐drying for immunohistochemical staining and various molecular biological analyses

Matsuo

Sugiyama

Okuyama

et al. 1999

Pathology International

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Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

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“…For secondary drying, the tissues in this study were subjected to a shelf temperature of +20 °C for 20 h and a collector temperature of –85 °C under vacuum of 0.01–0.02 mBar. Under these conditions, tumor samples were dehydrated to a water content of 1–2 % as determined by Karl Fischer titration, a standard method for measuring water content [19]. …”

Section: Methodsmentioning

confidence: 99%

Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage

et al. 2013

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Frozen tissue, a gold standard biospecimen, can yield well preserved nucleic acids and proteins after over a decade but is vulnerable to thawing and has substantial fiscal, spatial, and environmental costs. A long-term room temperature biospecimen storage alternative that preserves broad analytical utility can potentially empower tissue-based research. As there is scant data on the analytical utility of lyophilized brain tumor biospecimens, we evaluated lyophilized (freeze-dried) samples stored for 1 year at room temperature. Lyophilized tumor tissue processed into paraffin sections produced good histology. Yields of extracted DNA, RNA, and protein approximated those of frozen tissue. After 1 year, lyophilized samples yielded high molecular weight DNA that permitted copy number variation analysis, IDH 1 mutation detection, and MGMT promoter methylation PCR. A 27 % decrease in RIN scores over the 1 year suggests that RNA degradation was inhibited though incompletely. Nevertheless, RT-PCR studies on lyophilized tissue performed similarly to frozen tissue. In contrast to FFPE tissues where protein bands were absent or shifted to a lower molecular weight, lyophilized samples showed similar protein bands as frozen tissue on SDS-PAGE analysis. Lyophilized tissue performed similarly to frozen tissue for Western blots and enzyme activity assays. Immunohistochemistry of lyophilized tissue that were processed into FFPE blocks often required longer incubation times for staining than standard FFPE samples but generally provided robust antigen detection. This preliminary study suggests that lyophilization has promise for long-term room temperature storage while permitting varied tests; however, further work is required to better stabilize nucleic acids particularly RNA.

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Degradation of Macromolecules during Preservation of Lyophilized Pathological Tissues

Cited by 12 publications

References 16 publications

Freeze Substitution and Freeze Drying for Stable, Long-Term Preservation of Cytologic Specimens for Immunostaining

Freeze Substitution and Freeze Drying for Stable, Long-Term Preservation of Cytologic Specimens for Immunostaining

Preservation of pathological tissue specimens by freeze‐drying for immunohistochemical staining and various molecular biological analyses

Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage

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