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

Abstract

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.

Biomarker Discovery in Non–Small Cell Lung Cancer: Integrating Gene Expression Profiling, Meta-analysis, and Tissue Microarray Validation

“…Noteworthy is the inclusion of patient performance status, a parameter not annotated in previous datasets. Histopathologic review, fresh-frozen tissue handling, selection of representative tissue with high tumor cell content, and mRNA extraction were conducted using uniform and standardized protocols within the infrastructure of an established biobank and a diagnostic molecular pathology laboratory (20)(21)(22)(23). Thus, we believe that artifacts due to poor tissue quality and methodologic inconsistencies have been minimized.…”

“…All fresh-frozen tissue samples were collected using the same standardized protocol (20)(21)(22)(23), and each frozen section was reviewed by pathologists (P. Micke and J. Botling) to confirm that the sample contained representative tumor tissue.…”

The State of RT-Quantitative PCR: Firsthand Observations of Implementation of Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE)

“…Keeping tissue frozen until homogenization in a solution containing RNase inhibitors ensures consistent results by preventing inconsistent thawing of samples, which leads to differential RNA degradation [Botling et al, 2009;Huang et al, 2013;Kirschner et al, 2013]. Some labs remove tissue samples from -80 ° C storage, transport them on blue ice and then proceed to homogenize samples and extract the RNA using a wide variety of techniques, during which the samples may begin to thaw prior to RNA extraction.…”

Using measurements of muscle cell nuclear RNA with flow cytometry to improve assessment of larval condition of Walleye Pollock (Gadus chalcogrammus)

“…Neither freezing and thawing nor storage of frozen tissue affected RNA in muscle cell nuclei of larvae of Walleye Pollock in our study. For frozen human tissue, no signifi cant RNA degradation (as measured by gene expression and electropherograms) was detected after 16 h on ice (Micke et al, 2006), and another study showed that signifi cant RNA degradation did not begin until 30 min after thawing at room temperature (Botling et al, 2009). Although those studies did not measure the amount of RNA present, they do support our assertion that the protocol used in our study was adequate to preserve RNA because larvae were frozen quickly, thawed tissue was kept cool on ice, and the time from tissue thawing to analysis with fl ow cytometry was typically not longer than 5 h. Our results differ from the fi ndings of Theilacker and Shen (1993b) in that their study indicated that a cryoprotectant and acid were needed before freezing to stabilize RNA in brain cells of larvae of Walleye Pollock.…”