Temporary Ischemia Time Before Snap Freezing Is Important for Maintaining High-Integrity RNA in Hepatocellular Carcinoma Tissues.
Author(s): Zheng H, Tao YP, Chen FQ, Li HF, Zhang ZD, Zhou XX, Yang Y, Zhou WP
Publication: Biopreserv Biobank, 2019, Vol. , Page
PubMed ID: 31025876 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of cold ischemia duration and temperature on RNA yield and integrity in snap-frozen tissue.
Conclusion of Paper
RNA yields were comparable among tumor and matched normal adjacent specimens, regardless of ischemia time and temperature. RINs were comparable between tumor specimens maintained at 4°C and 24°C for ≤15 min but decreased after 15 min at 24°C and after 30 min at 4 °C. RINs in normal adjacent tissues decreased significantly beginning at 15 min for both temperatures. There were no significant differences in RIN between tumor and matched adjacent normal tissues stored at 4°C or 24°C when ischemia was ≤15 min, but RINs were higher in tumor than normal adjacent tissue specimens after 30 min ischemia at either temperature. Results were confirmed by gel electrophoresis analysis of 28S and 18S ribosomal RNA.
Studies
-
Study Purpose
This study investigated the effects of cold ischemia duration and temperature on RNA yield and integrity in snap-frozen tissue. Tissue specimens were collected from hepatocellular carcinoma patients that had not received neoadjuvant chemotherapy and/or radiotherapy before surgery. Tumor and normal adjacent tissues were divided into eight pieces and maintained at 4°C or 24°C for 15, 30, 60, or 120 minutes. Specimens were then divided into approximately 0.2 g (5 x 5 x 3 mm3) pieces, placed in a cryovial, snap-frozen in liquid nitrogen, and stored in the vapor phase of liquid nitrogen. Frozen specimens were ground with a mortar and pestle under liquid nitrogen and RNA extracted using the RNeasy Mini Kit. RNA quantity was measured by an unspecified method, RIN determined by bioanalyzer, and RNA integrity was assessed by gel electrophoresis.
Summary of Findings:
RNA yields were comparable among tumor and matched normal adjacent specimens, regardless of ischemia time and temperature. RINs were comparable between tumor specimens maintained at 4°C and 24°C for ≤15 min but decreased after 15 min at 24°C and after 30 min at 4 °C and continued to decrease significantly after 60 and 120 min at both temperatures (P<0.01, all). RINs in normal adjacent tissues decreased significantly after 15 min at both 4°C and 24°C (P<0.001) and continued to decrease after 60 and 120 min at both temperatures (P<0.01, all). There were no significant differences in RIN between tumor and matched adjacent normal tissues at 4°C or 24°C when ischemia was ≤15 min, but RINs were higher in tumor than normal adjacent tissue after 30 min ischemia at both temperatures (P<0.001) and after 60 min at 4°C (P<0.01) but RINs were comparable among tissue types after 60 and 120 min at 24°C (P>0.05, both). Results were confirmed by gel electrophoresis analysis of 28S and 18S ribosomal RNA with specimens of RIN >7 demonstrating two clear main bands, specimens with RIN between 4-7 resulted in reduced clarity and a blurry 28S band, and specimens with RIN <4 resulted in both bands disappearing or appearing as a smear.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Electrophoresis RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 4°C
24°C
Biospecimen Acquisition Cold ischemia time 15 min
30 min
60 min
120 min