RNA and DNA Integrity Remain Stable in Frozen Tissue After Long-Term Storage at Cryogenic Temperatures: A Report from the Ontario Tumour Bank.
Author(s): Kelly R, Albert M, de Ladurantaye M, Moore M, Dokun O, Bartlett JMS
Publication: Biopreserv Biobank, 2019, Vol. , Page
PubMed ID: 30762427 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of long-term storage of tissue specimens in vapor-phase liquid nitrogen (VPLN) on RNA yield, RNA purity, RNA integrity number (RIN), and DNA integrity.
Conclusion of Paper
The difference in RIN between the two assessments of replicate specimens was not correlated with storage duration. For specimens stored in VPLN >1 year, there was no correlation between storage duration and RNA yield, RNA purity, RIN, or DNA integrity number (DIN). Further, a specimen’s RIN was not predictive of DIN.
Studies
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Study Purpose
This study investigated the effects of long-term storage of tissue specimens in VPLN on RNA yield, RNA purity, RIN, and DNA integrity. Tumor and normal adjacent tissue specimens (bladder, breast, central nervous system, colon, endocrine, esophagus, stomach, head and neck, lung, ovary, rectum, kidney, soft tissue sarcoma, and uterus) from 87 cases were collected between 2005 and 2014 by five collection centers and immediately transported from the operating room to the pathology department. Specimens were snap-frozen by an unspecified method within 30 minutes of resection in 2 mL cryovials and stored in vapor-phase liquid nitrogen (VPLN). As part of an external quality assessment, specimens were shipped on dry ice to the processing site and RNA was extracted within one year of collection from ~100 mg of tissue using a standard chloroform-based method (prior to 2010) or using the RNeasy Mini Kit and Qiacube automatic extractor (2010 and later). RNA was extracted from additional specimens of the same tumors after storage in VPLN for up to 11 years. These specimens were incubated overnight at an unspecified temperature in three volumes of RNALater-Ice, diced, transferred to 2.5 mL of QIAzol Lysis Reagent, and homogenized using a tissue disruptor before RNA extraction using the RNeasy Mini Kit or the miRNeasy Mini Kit. RNA yield and purity were measured with a spectrophotometer and RINs determined by bioanalyzer. DNA was extracted from ~50mg of tissue from a subset of samples (n=20) using the DNeasy Blood and Tissue Kit. DNA integrity was assessed by DNA integrity number (DIN) using a TapeStation.
Summary of Findings:
The difference in RIN values between matched specimens extracted within a year and after up to 11 years in VPLN ranged from -7.5 to 6.3 (median change -0.3) with 69% of specimens (59/85) exhibiting a difference in RIN ≤2.0. The difference in RIN did not correlate with storage duration (r=0.01074, P=0.3193). For specimens stored in VPLN >1 year, there was no correlation between storage duration and RNA yield or purity (r= -0.0201, P=0.8533 and r= -0.1071, P=0.3235; respectively) or RIN (r=0.0514, P=0.6346). There was no significant correlation between storage duration in VPLN and DNA integrity (r=0.250, P=0.287) or between RIN and DIN (r=0.2362, P=0.3147). Further, a specimen’s RIN was not predictive of DIN (r=0.2362, P=0.3147).
Biospecimens
- Tissue - Bladder
- Tissue - Breast
- Tissue - Colorectal
- Tissue - Esophagus
- Tissue - Stomach
- Tissue - Other
- Tissue - Head and Neck
- Tissue - Lung
- Tissue - Kidney
- Tissue - Ovary
- Tissue - Uterus
- Tissue - Nerve
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Sarcoma
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer DNA Automated electrophoresis/Bioanalyzer RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration < 1 year
1.7 to 11.4 years