Influence of Freeze-Thaw Cycles on RNA Integrity of Gastrointestinal Cancer and Matched Adjacent Tissues.
Author(s): Hu Y, Han H, Wang Y, Song L, Cheng X, Xing X, Dong B, Wang X, Chen M, Zhang L, Ji J
Publication: Biopreserv Biobank, 2017, Vol. 15, Page 241-247
PubMed ID: 28170288 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of freeze-thaw cycling on RNA integrity and morphology in matched gastrointestinal tumor and normal adjacent tissue.
Conclusion of Paper
Mean RIN scores decreased significantly after one freeze-thaw cycle in all specimens except liver specimens (cancer and adjacent tissues) and additional degradation was observed after two freeze/thaw cycles. Overall, normal adjacent tissues demonstrated comparatively more degradation than matched cancer tissue specimens. While repeated freeze-thaw cycles did not adversely affect morphology for any of the specimens analyzed, all specimens evaluated for morphology were first snap frozen in liquid nitrogen in the absence of a cryoprotectant and preserved in RNAlater prior to OCT embedding and histological analysis.
Studies
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Study Purpose
This study investigated effects of freeze-thaw cycling on RNA integrity and morphology in matched gastrointestinal tumor and normal adjacent tissue. Specimens were collected from 46 patients with various gastrointestinal cancers (24 gastric, 5 liver, 5 colon, 5 rectal, and 7 pancreatic) and immediately snap-frozen in liquid nitrogen. Specimens were then divided into similarly sized pieces (4 x 4 x 4 mm) and one was placed in RNAlater on ice (T0); one was immediately frozen at -80°C for 30 minutes, thawed at room temperature for 10 minutes, then placed in RNAlater (T1); and a third piece went through two freeze-thaw cycles before being placed in RNAlater (T2). Specimens were ground to a powder in liquid nitrogen, RNA was extracted with Trizol, and RIN was determined using a bioanalyzer. Additional sections were embedded in optimal cutting temperature compound (OCT), frozen at -80°C, cut into 10 mm-thick sections on a cryostat, H&E stained, and examined for morphology by two pathologists.
Summary of Findings:
RIN values were greater than 6 for all tissues snap-frozen immediately and placed in RNAlater (T0), with the exception of one of the seven (1/7) pancreatic cancer specimens and five of the seven (5/7) normal adjacent specimens. Mean RIN scores decreased significantly after one freeze-thaw cycle (T1) as RIN scores declined to less than 6 in 7 of the 24 gastric cancer and 20 of the 24 noncancerous gastric specimens, 1 of the 5 colon cancer and 4 of the 5 noncancerous colon specimens, and all 5 of the noncancerous rectal specimens (P<0.05, all). Conversely, RIN was not altered by one freeze-thaw cycle in liver cancer and adjacent tissues. Additional degradation associated with two freeze/thaw cycles (T2) was observed in 9 of the 24 gastric cancer and 23 of the 24 noncancerous gastric specimens. Overall, normal adjacent tissues demonstrated comparatively more degradation than matched cancer tissue specimens. While the authors conclude that repeated freeze-thaw cycles did not adversely influence morphology for any of the specimens, all specimens evaluated for morphology were first snap-frozen in liquid nitrogen in the absence of a cryoprotectant and preserved in RNAlater prior to OCT embedding and histological analysis.
Biospecimens
Preservative Types
- Frozen
- RNAlater
- OCT
Diagnoses:
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer Morphology H-and-E microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location stomach
liver
colorectal
pancreas
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles