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

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.

[1]  Hans-Georg Rammensee,et al.  Moderate degradation does not preclude microarray analysis of small amounts of RNA. , 2003, BioTechniques.

[2]  Charles Auffray,et al.  Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces , 2005, Nucleic acids research.

[3]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[4]  D. Goldstein,et al.  Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization , 2005, Laboratory Investigation.

[5]  Matthias Nees,et al.  Impact of pre‐analytical handling on bone marrow mRNA gene expression , 2004, British journal of haematology.

[6]  David A Jones,et al.  Preservation of RNA for Functional Genomic Studies: A Multidisciplinary Tumor Bank Protocol , 2001, Modern Pathology.

[7]  Donna Neuberg,et al.  Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays , 2004, BMC Genomics.

[8]  Fredrik Ponten,et al.  A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissection and pressure catapulting , 2004, The Journal of pathology.

[9]  Karl Kornacker,et al.  Chipping away at the chip bias: RNA degradation in microarray analysis , 2003, Nature Genetics.

[10]  J. Crawford,et al.  Pathology as the enabler of human research , 2005, Laboratory Investigation.

[11]  F. Radvanyi,et al.  Gene expression analysis by real-time reverse transcription polymerase chain reaction: influence of tissue handling. , 2004, Analytical biochemistry.

[12]  T. Daly,et al.  Precision profiling and components of variability analysis for Affymetrix microarray assays run in a clinical context. , 2005, The Journal of molecular diagnostics : JMD.

[13]  K. Creek,et al.  RNA degradation in human breast tissue after surgical removal: a time-course study. , 2004, Experimental and molecular pathology.

[14]  M. Sherman,et al.  Cervical Tissue Collection Methods for RNA Preservation: Comparison of Snap-frozen, Ethanol-fixed, and RNAlater-fixation , 2006, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[15]  S. Bonin,et al.  A Novel Fixative Improves Opportunities of Nucleic Acids and Proteomic Analysis in Human Archive's Tissues , 2006, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[16]  D. Ginzinger,et al.  Effect of Duration of Fixation on Quantitative Reverse Transcription Polymerase Chain Reaction Analyses , 2002, Modern Pathology.

[17]  J Quackenbush,et al.  Effects of ischemia on gene expression. , 2001, The Journal of surgical research.

[18]  F. Pontén,et al.  Zinc-Based Fixative Improves Preservation of Genomic DNA and Proteins in Histoprocessing of Human Tissues , 2003, Laboratory Investigation.

[19]  Jeffrey Wilusz,et al.  The highways and byways of mRNA decay , 2007, Nature Reviews Molecular Cell Biology.

[20]  W. Sommergruber,et al.  Laser Capture Microdissection of Epithelial Cancers Guided by Antibodies Against Fibroblast Activation Protein and Endosialin , 2006, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[21]  C. Dumur,et al.  Assessing the Impact of Tissue Devitalization Time on Genome-wide Gene Expression Analysis in Ovarian Tumor Samples , 2008, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[22]  I. Ferrer,et al.  DNA Chip Technology in Brain Banks: Confronting a Degrading World , 2004, Journal of neuropathology and experimental neurology.

[23]  F. Pontén,et al.  Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens , 2006, Laboratory Investigation.

[24]  Michael W Pfaffl,et al.  RNA integrity and the effect on the real-time qRT-PCR performance. , 2006, Molecular aspects of medicine.

[25]  M. Ellis,et al.  Development and validation of a method for using breast core needle biopsies for gene expression microarray analyses. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[26]  F. Pontén,et al.  Laser-assisted cell microdissection using the PALM system. , 2005, Methods in molecular biology.