Compared DNA and RNA quality of breast cancer biobanking samples after long-term storage protocols in - 80 °C and liquid nitrogen.
Author(s): Babel M, Mamilos A, Seitz S, Niedermair T, Weber F, Anzeneder T, Ortmann O, Dietmaier W, Brochhausen C
Publication: Sci Rep, 2020, Vol. 10, Page 14404
PubMed ID: 32873858 PubMed Review Paper? No
Purpose of Paper
This paper compared the effects of storage of matched breast tumor tissue specimens at -80°C to vapor-phase liquid nitrogen (VPLN) on RNA and DNA integrity as well as tissue morphology.
Conclusion of Paper
Mean RNA integrity numbers (RINs) were significantly higher for RNA extracted from tissue specimens stored in VPLN than those stored at -80°C and while the mean DNA integrity number (DIN) was higher in DNA from specimens stored in VPLN than at -80°C, the difference did not reach statistical significance. Specimens stored at -80°C were more likely to have significantly abnormal nuclei, loss of detection of cell–cell borders, nuclei with abnormal chromatin-distribution, and more diffuse desmosomes with poor contrast to the cytoplasm than specimens stored in VPLN.
Studies
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Study Purpose
This study compared the effects of storage at of matched breast tumor tissue specimens -80ºC to vapor-phase liquid nitrogen (VPLN) storage on RNA and DNA integrity and tissue morphology. Tumor specimens were collected by surgical resection from 16 breast cancer patients (details not provided), divided, immediately snap-frozen in liquid nitrogen, and half was stored at -80ºC and the other half was stored in VPLN. After storage for 10.0-12.2 y, specimens were divided into two parts on a dry ice-cooled, sterile cutting board. One half was put into 4% formalin for 24 h at room temperature, paraffin-embedded, and 5 μm-thick sections were prepared and H&E-stained in an automated processor. The other half was sectioned in 10 μm-thick sections in a cryostat for nucleic acid extraction and electron microscopy analysis. RNA was extracted from 5-20 sections (~30 mg) using the RNeasy Mini Kit and DNA was extracted from 5-10 sections (~20 mg) with the QIamp DNA Micro Kit. RIN and DIN values were determined using a TapeStation. Three 2×2×2 mm pieces from each frozen specimen were placed in Karnowsky’s fixative for at least 72 h at room temperature, embedded into Epon in an automated processor and ultra-thin sections (60 nm thickness) were made with a microtome which were analyzed using a transmission electron microscope.
Summary of Findings:
Mean RINs were significantly higher in RNA from tissue specimens stored in VPLN than those stored at -80°C (8.59 versus 7.14, P=0.0024). Further, 93.75% of specimens stored in VPLN had RNA with RINs ≥6.0 compared to only 81.5% of RNA from specimens stored at -80°C and 81.25% of VPLN-stored specimens had RNA with RINs ≥8.0 but only 31.25% of specimens stored at -80°C had RNA with RIN values of ≥8.0. The mean DIN value was higher in DNA from specimens stored in VPLN than at -80°C (6.99 versus 7.42) but the difference did not reach statistical significance (P=0.21). Morphology was better preserved in VPLN-stored specimens than those stored at -80°C with significantly abnormal nuclei observed in 2 of the 16 specimens stored in VPLN compared to 11 of 16 specimens stored at -80°C. Transmission electron microscopic analyses revealed incomplete or no detectable cell–cell borders, abnormal chromatin distribution, and more diffuse desmosomes with poor contrast to the cytoplasm in tumor cell specimens stored at -80°C compared to those stored in VPLN.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer Morphology Electron microscopy Morphology H-and-E microscopy RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature -80ºC
Vapor-phase liquid nitrogen