Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue.

Author(s): Botling J, Edlund K, Segersten U, Tahmasebpoor S, Engström M, Sundström M, Malmström PU, Micke P

Publication: Diagn Mol Pathol, 2009, Vol. 18, Page 44-52

PubMed ID: 19214109 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effect of thaw time (0 min - 16 h) on RNA integrity and gene expression in tonsil frozen in the presence or absence of RNA later.

   Summary of Findings:

   Samples frozen in the absence of RNA later first displayed a change in RNA integrity number (RIN) after a 30 min thaw duration at room temperature. Untreated frozen samples degraded rapidly with 28S and 18S ribosomal peaks becoming less distinct before disappearing completely between 6 h and 16h. Real-time qRT-PCR data first reflected altered transcripts of FOS, BCL-2 and TGFB1 in specimens thawed for 45 min. The more than 10 fold increase in FOS and BCL-2 was largely due to a change in cycle threshold values obtained for the normalizer gene GAPDH. The use of a shorter GAPDH amplicon (75 vs 174 bp) partially attenuated the effect as did a change in normalizer gene to HPRT1. In contrast, no effect of thaw time on RNA integrity or gene expression was observed in the RNA later treated frozen tissue specimens.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Frozen
   • RNAlater

   Diagnoses:

   • Not specified

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen RNAlater
   Real-time qRT-PCR Specific	Targeted nucleic acid	TGFB FOS HIF1A PCNA BCL2 HPRT1 GAPDH
   Storage	Thaw duration	0 5 min 30 min 45 min 1 h 3 h 6 h 16 h
   Real-time qRT-PCR Specific	Length of gene fragment	75 bp 174 bp

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2. Study Purpose

   The purpose of this study was to determine the effect of multiple thaw cycles (1- 6 cycles) on RNA integrity in frozen tonsil specimens stored in the presence or absence of RNAlater.

   Summary of Findings:

   Minimal degradation as determined by a decrease in RNA integrity number was first observed in samples frozen without RNAlater after 6 thaw cycles. In this study, 6 thaw cycles corresponded to a total thaw time of 30 minutes, the thaw time found to decrease in RNA integrity. No change in integrity of samples was found in samples frozen in the presence of RNAlater. The authors conclude that freeze thaw cycles are not detrimental to RNA integrity in tonsil specimens as long as total thaw time remains under 30 minutes.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • RNAlater
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Freeze/thaw cycling	1 cycle 3 cycles 6 cycles
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen RNAlater

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