Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Optimized Biobanking Procedures for Preservation of RNA in Tissue: Comparison of Snap-Freezing and RNAlater-Fixation Methods.

Author(s): Hentze JL, Kringelbach TM, Novotny GW, Hamid BH, Ravn V, Christensen IJ, Høgdall C, Høgdall E

Publication: Biopreserv Biobank, 2019, Vol. , Page

PubMed ID: 31618057 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared preservation of tumor tissue by snap-freezing and RNAlater and investigated the effects of cold ischemia time on RNA integrity numbers (RINs) and HPRT1 and B2M levels as determined by real-time qRT-PCR. Specimens from six female patients (age 35–70 y) undergoing operation for malignant gynecological disease were obtained from tumors at 0, 15, 30, 45, 60, 90, 120, and 180 minutes after surgery. Each specimen was divided into three pieces and formalin-fixed paraffin-embedded for H&E staining; snap-frozen in dry-ice cooled isopentane and then stored at -80°C; or preserved in RNAlater at 4°C for ≥24 hours and then excess RNAlater removed before storage at -80°C. The degree of tissue necrosis and tumor percentage was established from H&E-stained sections by a pathologist. Snap-frozen and RNAlater-preserved specimens were stored 5–7 years before 30 mg pieces were obtained by cutting specimens on a metal plate over dry ice, homogenized in cold lysis buffer, and stored for up to a year at -80°C until RNA isolation with the RNeasy Mini Kit. Purified RNA was stored at -80°C for up to 2 years. RNA concentrations were measured using Nano-Drop and RINs determined by bioanalyzer. Expression levels of B2M and HPRT1 were measured by real-time qRT-PCR.

   Summary of Findings:

   Intra-patient variation in RINs was observed but there was no significant difference based on cold ischemia time (P=0.91) or between RNAlater-preserved and snap-frozen specimens (P=0.17). As expected, specimens with a high RIN had lower Ct values than those with a lower RIN. RINs were correlated with HPRT1 levels (R=0.92 for snap-frozen specimens and R=0.62 for RNAlater specimens) but correlation with RIN was lower with B2M levels (R=0.26 for snap-frozen specimens and R=0.64 for RNAlater specimens. A trend toward higher RNA integrity (more amplifiable copies) was seen in RNAlater-preserved tissue compared to snap-frozen tissue when real-time qRT-PCR data for the two genes were analyzed separately (B2M: P=0.051, HPRT1: P=0.147). Combining the data for the two genes revealed that RNAlater was significantly better at preserving RNA than snap-freezing (P=0.03). There was no reduction in gene expression levels with increased cold ischemia time (P=0.99 for both B2M and HPRT1).

   Biospecimens

   • Tissue - Ovary

   Preservative Types

   • Frozen
   • RNAlater
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0 min 15 min 30 min 45 min 60 min 90 min 120 min 180 min
   Real-time qRT-PCR Specific	Targeted nucleic acid	B2M HPRT1
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen RNAlater

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