Department of Pathology and Laboratory Medicine Neuropathology, UCLA School of Medicine, Los Angeles, CA 90095, USA.
J Neurooncol. 2013 Jul;113(3):365-73. doi: 10.1007/s11060-013-1135-1. Epub 2013 May 3.
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.
冷冻组织是一种金标准生物标本，在经过十多年的时间后仍能获得保存良好的核酸和蛋白质，但易受解冻影响，且具有巨大的财政、空间和环境成本。一种长期室温下生物标本储存的替代方法，如果能保持广泛的分析适用性，可能会为基于组织的研究提供支持。由于关于冻干脑肿瘤生物标本分析适用性的数据很少，我们评估了在室温下储存 1 年的冻干（冷冻干燥）样本。经过冻干处理的肿瘤组织制成石蜡切片后，组织学表现良好。提取的 DNA、RNA 和蛋白质的产量与冷冻组织相近。经过 1 年，冻干样本产生了高分子量的 DNA，可用于进行拷贝数变异分析、IDH1 突变检测和 MGMT 启动子甲基化 PCR。在 1 年内，RIN 评分下降了 27%，这表明 RNA 降解虽受到抑制，但并不完全。尽管如此，冻干组织的 RT-PCR 研究与冷冻组织相似。与 FFPE 组织相比，冻干样本的蛋白质条带不存在或迁移到较低分子量，SDS-PAGE 分析显示，冻干样本与冷冻组织的蛋白质条带相似。在 Western blot 和酶活性测定中，冻干组织的表现与冷冻组织相似。将冻干组织加工成 FFPE 块进行免疫组织化学染色时，与标准 FFPE 样本相比，染色所需的孵育时间通常更长，但通常可以提供强大的抗原检测。这项初步研究表明，冻干技术在允许进行各种测试的同时，具有在室温下长期储存的潜力；然而，需要进一步的工作来更好地稳定核酸，特别是 RNA。
