Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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RNA-Integrity and 8-Isoprostane Levels Are Stable in Prostate Tissue Samples Upon Long-Term Storage at -80°C.

Author(s): Dybos SA, Brustad ÅW, Rolfseng T, Kvam S, Olsen OE, Halgunset J, Skogseth H

Publication: Biopreserv Biobank, 2020, Vol. , Page

PubMed ID: 32865438 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of storage duration (2-15 y) at -80°C and RNA extraction kit used on RNA yield, 260/280 ratio, RNA Integrity Numbers (RINs), and levels of 8-isoprostane in snap-frozen prostate tissue specimens. Prostate tissue was obtained from 97 patients undergoing radical prostatectomies between 2003 and 2016. A 2 mm-thick slice was cut perpendicularly to the urethra within 10–15 minutes after resection, snap-frozen between two metal plates precooled with liquid nitrogen, and stored in sealed zip bags at -80°C until analysis in 2018. To investigate the effects of storage duration and compare RNA extraction kits, two 3-mm core specimens were collected from tumor-free areas of 65 tissue slices collected during 2005–2007 (n=27) and in 2016 (n=38) using a high-speed drill unit while keeping the specimen frozen on an aluminum block continuously cooled by contact with liquid nitrogen. RNA was extracted using the miRNeasy Mini Kit for one core and the mirVana miRNA Isolation Kit from the remaining core. RNA yield and 260/280 ratio were determined using a NanoDrop Spectrophotometer and RINs were assigned by a bioanalyzer. To investigate the effect of storage duration on levels of 8-isoprostane, one 3 mm core from each of 32 tissue slices collected from 2003-2016 were mechanically homogenized in an EDTA/butylated hydroxytoluene/phosphate buffer solution, lipids were eluted on affinity columns, and 8-isoprostane levels were measured by ELISA.

   Summary of Findings:

   There was no statistically significant difference in mean RNA yield between tissue extracted with the mirVana Kit compared to the miRNeasy Kit in specimens stored for 11-13 y (31.0 mg and 25.4, respectively) or for the specimens stored for 2 y (32.0 mg and 28.1 mg, respectively). The mean 260/280 ratios were comparable for RNA from tissue stored for 11-13 y and those stored for 2 y when extracted using the mirVana Kit (2.04 and 2.03, respectively) and the miRNeasy Kit (2.11 and 2.08, respectively). The mean RIN for RNA extracted with the mirVana Kit was higher than for RNA extracted using the miRNeasy Kit in specimens stored for 11-13 y (9.4 versus 6.2, P<0.001) and in specimens stored for 2 y (9.7 versus 7.1, P<0.001). RINs were significantly higher for specimens stored for 11-13 y than for those stored for 2 y when extracted with the miRNeasy Kit (7.1 versus 6.2, P<0.001) but the differences were not statistically significant when RNA was extracted with the mirVana Kit (9.4 versus 9.7, P=0.083). Overall, average RIN was higher in specimens extracted with the mirVana Kit compared to the miRNeasy Kit (9.6 versus 6.7, P<0.001). Levels of 8-isoprostane were unaffected in prostate tissue stored for up to 15 y.

   Biospecimens

   • Tissue - Prostate

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer
   Lipid	ELISA

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy Mini Kit mirVana miRNA Isolation Kit
   Storage	Storage duration	2 y 11-13 y 2-15 y

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