Biobanking of Fresh-frozen Human Colon Tissues: Impact of Tissue Ex-vivo Ischemia Times and Storage Periods on RNA Quality.
Author(s): Bao WG, Zhang X, Zhang JG, Zhou WJ, Bi TN, Wang JC, Yan WH, Lin A
Publication: Ann Surg Oncol, 2012, Vol. 20(5), Page 1737-44
PubMed ID: 22711177 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to assess the extent to which RNA integrity is affected by the duration and temperature of cold ischemia (a post excision delay prior to preservation) and the duration of frozen storage.
Conclusion of Paper
RNA integrity, assessed by gel electrophoresis and RNA integrity number (RIN), was not affected by a cold ischemia time of up to 4 h at room temperature or on wet ice, or by storage at -80 degrees C for up to 40 months. However, significant differences were observed for RNA yield and RIN among case-matched cancerous and normal adjacent specimens. Cancerous specimens had significantly higher RINs and RNA yields (0.39 versus 0.24 ug) compared to normal adjacent counterparts (p<0.001). The authors hypothesize that RIN is influenced by the proportion of connective tissue within a sample, and this differed between the cancerous and normal adjacent specimens investigated.
Studies
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Study Purpose
The purpose of this study was to determine if RNA integrity is affected by the duration or temperature of cold ischemia time. Colon cancer and normal adjacent specimens were collected via surgical resection from 5 patients and subjected to a delay at room temperature or on wet ice prior to snap freezing in liquid nitrogen. All specimens were stored at -80 degrees C prior to analysis.
Summary of Findings:
Evidence of RNA degradation was not detectable by electropherogram or microchip gel electrophoresis among cancerous colon specimens subjected cold ischemia times ranging from 30 min to 4 h either at room temperature or on wet ice. RINs were similarly unaffected by cold ischemia time and temperature, with comparable RINs reported for case-matched specimens that were immediately snap-frozen (RIN 8.8) and those subjected to a cold ischemia time of 4 h (RIN 8.3-8.5).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 30 min
1 h
2 h
4 h
Storage Storage temperature Room temperature
Wet ice
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Study Purpose
The purpose of this study was to determine if RNA integrity is altered by prolonged frozen storage. Potential effects associated with cold ischemia time were also explored. Colon cancer and normal adjacent specimens collected via surgical resection from 51 patients were snap-frozen in liquid nitrogen and stored in a -80 degrees C freezer for 15-40 months prior to analysis.
Summary of Findings:
While 80% of the total specimens examined had an RIN of 7 or greater, the percentage of specimens meeting this threshold was significantly higher for cancerous specimens (94%) compared to normal adjacent specimens (67%; p<0.001). In comparison to case-matched normal adjacent specimens, cancerous specimens also had significantly higher RNA yields (0.39 versus 0.24 ug) and RINs, with differences in RIN ranging between 0 and 9 (p<0.001). While a potential correlation between RIN and recorded cold ischemia time were evaluated for 39 specimens, a significant relationship was not observed, although RINs were reported to be 7 or greater for the sample set. There was no clear relationship between RIN and the duration of frozen storage, and the authors conclude that storage for up to 40 months at -80 degrees C does not negatively affect RNA integrity. The authors hypothesize that RIN is influenced by tissue composition, as the cancerous specimens analyzed had a epithelial cancer cell content of 70% or greater while normal adjacent specimens were rich in connective tissue.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 15 months or less
16-20 months
21-25 months
26-40 months
Biospecimen Acquisition Cold ischemia time 15 min or less
16-30 min
31-60 min
Biospecimen Aliquots and Components Biospecimen heterogeneity Rich in connective tissue
Rich in epithelial cells