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Impact of Thawing on RNA Integrity and Gene Expression Analysis in Fresh Frozen Tissue

Botling, Johan MD, PhD*; Edlund, Karolina BSc*; Segersten, Ulrika PhD; Tahmasebpoor, Simin BSc*; Engström, Mats MD, PhD; Sundström, Magnus PhD*; Malmström, Per-Uno MD, PhD; Micke, Patrick MD*

Diagnostic Molecular Pathology: March 2009 - Volume 18 - Issue 1 - pp 44-52
doi: 10.1097/PDM.0b013e3181857e92
Original Articles

Biobanks of fresh, unfixed human tissue represent a valuable source for gene expression analysis in translational research and molecular pathology. The aim of this study was to evaluate the impact of thawing on RNA integrity and gene expression in fresh frozen tissue specimens. Portions of snap frozen tonsil tissue, unfixed or immersed in RNAlater, were thawed at room temperature for 0 minute, 5 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, and 16 hours before RNA extraction. Additionally, tonsil tissue underwent repetitive freezing and thawing cycles. RNA integrity was analyzed by microchip gel electrophoresis and gene expression by quantitative real-time polymerase chain reaction for selected genes (FOS, TGFB1, HIF1A, BCL2, and PCNA). Minimal RNA degradation was detected after 30 minutes of thawing in unfixed samples. This degradation was accompanied by relevant changes in gene expression for FOS and BCL2 at 45 minutes. Modified primer design or the use of different housekeeping genes could not rectify the changes for FOS. Repetitive thawing cycles had similar effects on RNA integrity. The incubation of the tissue in RNAlater efficiently prevented RNA degradation. In conclusion, degradation of RNA in frozen tissue occurs first after several minutes of thawing. Already minimal decrease in RNA quality may result in significant changes in gene expression patterns in clinical tissue samples.

Departments of *Genetics and Pathology


Otorhinolaryngology, University Hospital, Uppsala, Sweden

The Fresh Tissue Biobank at the Department of Pathology, Uppsala University Hospital, was supported by the National Biobank Platform funded by Wallenberg Consortium North and SWEGENE.

Johan Botling and Karolina Edlund contributed equally to the study.

Reprints: Patrick Micke, MD, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE 751 85 Uppsala, Sweden (e-mail:

© 2009 Lippincott Williams & Wilkins, Inc.