Evaluation of a standardized protocol for processing adrenal tumor samples: preparation for a European adrenal tumor bank.

Author(s): Johnsen IK, Hahner S, Brière JJ, Ozimek A, Gimenez-Roqueplo AP, Hantel C, Adam P, Bertherat J, Beuschlein F

Publication: Horm Metab Res, 2010, Vol. 42, Page 93-101

PubMed ID: 19882499 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if cold ischemia time (time at room temperature prior to preservation) affects DNA integrity, RNA expression, protein expression levels, phosphorylation status, and enzymatic activity in adrenal specimens that were snap-frozen or pretreated with RNAlater overnight at 4 degrees C prior to storage at -80 degrees C.

   Summary of Findings:

   DNA integrity as determined by electrophoresis and PCR amplification of a 5.3 kb MC2-receptor amplicon, was not affected by the method of preservation or cold ischemia durations of up to 60 min at room temperature. While RNA integrity, as determined by a Bioanalyzer, did not differ significantly among snap-frozen and RNAlater preserved specimens, HPRT expression was significantly albeit modestly elevated in RNAlater preserved specimens. P450 c17 and COMT gene expression, markers of adrenal function, significantly varied after 60 min of ischemia compared to 15 min controls in both snap-frozen and RNAlater preserved specimens. Interestingly, differentially preserved specimens differed significantly from one another after 15 and 60 min of ischemia, with higher HPRT, P450 c17, COMT expression observed among RNAlater preserved specimens compared to those snap-frozen. Protein recovery and 3beta-HSD and chromogranin A levels did not differ with respect to preservation method. Although total ERK was not affected, phosphorylated ERK progressively decreased with cold ischemia (at room temperature) (15 versus 30 min); although statistical significance was only observed among specimens snap-frozen in liquid nitrogen, RNAlater preserved specimens exhibited a nonsigificant decrease of comparable magnitude. Enzymatic cytochrome c reductase and cytochrome c oxidase activities were reduced in RNAlater pretreated specimens compared to those that were snap-frozen.

   Biospecimens

   • Tissue - Adrenal Gland

   Preservative Types

   • Frozen
   • RNAlater

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   DNA	Electrophoresis
   DNA	PCR
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	15 min 30 min 60 min
   Biospecimen Preservation	Type of fixation/preservation	RNAlater Snap frozen
   PCR Specific	Targeted nucleic acid	MC2-receptor
   Real-time qRT-PCR Specific	Targeted nucleic acid	Beta-actin Hypoxanthine guanine phospho ribosyl-transferase
   Western blot Specific	Targeted peptide/protein	ERK Phospho-ERK 3beta-HSD Beta-actin

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2. Study Purpose

   The purpose of this study was to determine if thaw duration at room temperature of a frozen biospecimen affects RNA and protein expression levels, and protein phosphorylation status in adrenal specimens that were snap-frozen or pretreated with RNAlater overnight at 4 degrees C prior to storage at -80 degrees C.

   Summary of Findings:

   Thawing at room temperature of snap frozen tissue specimens prior to extraction resulted in a time-dependent decrease in detectable levels of both HPRT and beta-actin transcripts, effects that were not observed in RNAlater preserved specimens. Phosphorylated but not total ERK decreased with room temperature thaw duration in snap-frozen but not RNAlater pretreated specimens.

   Biospecimens

   • Tissue - Adrenal Gland

   Preservative Types

   • Frozen
   • RNAlater

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	30 min
   Biospecimen Preservation	Type of fixation/preservation	RNAlater Snap frozen
   Storage	Thaw duration	0 h 1 h 3 h
   Real-time qRT-PCR Specific	Targeted nucleic acid	HRT Beta-actin
   Western blot Specific	Targeted peptide/protein	Total ERK Phospho-ERK

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