Opitz et al. BMC Medical Genomics 2010, 3:36 http://www.biomedcentral.com/1755-8794/3/36 RESEARCH ARTICLE Open Access Impact of RNA degradation on gene expression profiling Lennart Opitz1, Gabriela Salinas-Riester1, Marian Grade2, Klaus Jung3, Peter Jo2, Georg Emons2, B Michael Ghadimi2, Tim Beißbarth3*†, Jochen Gaedcke2† Abstract Background: Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis. Methods: To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time- dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences. Results: The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5′ end. Conclusions: Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5′ end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings. Background considered a prerequisite for high-throughput analysis. High-throughput microarray technology is ideally suited Nevertheless, RNA degradation is a critical problem in all for analyzing thousands of genes in a single experiment experimental settings and for clinical samples in particu- and allows a better understanding of the molecular lar. Additionally, the investigation of clinical samples mechanisms of cancer development and progression. poses a second problem. Due to the heterogeneity of Gene expression arrays have a profound influence on the tumor samples, a high number of patients is needed for development of new diagnostic and therapeutic strate- statistical analysis. There is thus an ongoing debate as to gies, such as the prediction of prognosis in breast cancer how far gene expression results are affected by various [1] or the response to a preoperative radio-chemotherapy degrees of degradation [3-6] and to what extent of degra- (RT/CT) in rectal cancer [2]. High-quality RNA is dation the tissue samples with poor RNA quality can be included in an analysis. * Correspondence: [email protected] Degradation of RNA itself is a physiological process † Contributed equally during the cell cycle to regulate RNA-dependent 3 Department Medical Statistics, University Medicine Göttingen, mechanisms. Many intracellular enzymes, such as ribo- Humboldtallee 32, 37073 Göttingen, Germany Full list of author information is available at the end of the article nucleases, (endo- and exonucleases), as well as other © 2010 Opitz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Opitz et al. BMC Medical Genomics 2010, 3:36 Page 2 of 14 http://www.biomedcentral.com/1755-8794/3/36 cofactors, are involved and exhibit prevalent activity in chemotherapy. These patients were treated within the all organisms of life [7]. The multiplicity of functions CAO/ARO/AIO-04 trial (EudraCT-Number: 2006- that characterize ribonucleases in eukaryotes underlines 002385-20), biopsies were taken according to the guide- the key importance of mechanisms that specifically not lines set by the Local Ethical Review Board (application only target and degrade RNAs but also function in number: 9/8/08) and patients gave written consent. Due to RNA-processing reactions and presumably enhance the the rigid rectoscopy, biopsies were stored in RNAlater overall efficiency of degradation pathways [8,9]. (Qiagen, Hilden, Germany) within 10 to 20 seconds after Although these processes were well investigated in the removal and subsequently kept at 5°C for 24 hours fol- past and the level of knowledge is increasing steadily, lit- lowed by storage at -20°C. Extraction of nucleic acids was tle is known as to how far the mechanisms can be trans- carried out four months prior to the following experi- lated into cells that have been taken out of the organism ments and was performed using TRIZOL (Invitrogen, as is done when biopsies are taken. Carlsbad, CA) following standard procedures as previously Moreover, beside RNA-degrading enzymes and cofac- described [15] (details can be found at http://www.riedlab. tors, tissue-specific factors such as the extent of necrosis nci.nih.gov/protocols.asp) and stored at -80°C. and storage conditions have to be considered to avoid degradation of RNA. In the past, liquid nitrogen was RNA Degradation and Quality Control considered to be the standard method for archiving tis- Heat-induced degradation was carried out at 60°C on a sue samples. This, however, poses numerous logistical thermal block (Eppendorf Thermomixer). For each of problems surrounding the provision of nitrogen and the the three patients, four different time points were transportation of frozen tissue samples. This is essen- defined for the analysis of degradation state, namely tially a problem for clinical trials, in which patients are 0:00 h; 1:45 h; 2:30 h; and 3:15 h respectively. Time frequently recruited in non-university hospitals where points and degradation temperature were chosen based access to liquid nitrogen cannot be guaranteed. There- on preliminary test results (data not shown) revealing fore, preserving liquids have been developed and studied that no and very low degradation occurred at room tem- with respect to RNA stability [10-12]. perature and 45°C, respectively. To assess RNA quality, different methods have been After thermal degradation, total RNA was checked for applied [5,13]. The electrophoretic-based generation of a quantity, purity and integrity of the 18 S and 28 S ribo- RNA integrity number (RIN) [14] using Agilent’s Bioana- somal bands by capillary electrophoresis. RNA degrada- lyzer provides a user-independent method of reproduci- tion was assessed using the Agilent 2100 Bioanalyzer bly assessing degradation of RNA. In the recently following the manufacturer’s standard protocol. In brief, published literature comparing different levels of RNA after loading RNA Nano LabChip with the gel-dye-mix, integrity, the RIN has frequently been used, thus allowing each of the 12 measuring chambers was filled with RNA easy comparison of the postulated values that were indi- (concentration between 200-300 ng/μl) and the provided cated as thresholds for considering good and poor RNA marker. The measurement was then carried out by the quality [4,5]. Within these publications, tissue was treated Bioanalyzer and a separate RNA integrity number as with different temperature levels to achieve degradation. well as the correlating electropherogram and a gel-like Interestingly, the influence of similar temperature levels image was provided by the software for each of the sam- resulted in very different degradation results. Further- ples. Each RNA sample was split between four different more this pre-isolation degradation more or less mimics tubes. RNA quality was rated according to the RNA a prolonged time to storage and initiates the complex integrity number (RIN). Measurement of RIN was car- process of cell hypoxia and consecutive cell death. ried out prior to the array hybridization without freezing Here in this study, we aim to assess the influence of and thawing again. RNA degradation on gene expression and to analyze the systematic effect that is observable when using RNA with Microarrays different RINs. Therefore, we performed gene expression Microarrays were done using the “Low RNA Input lin- analysis of tissues from tumor biopsies of several patients ear Amplification Kit Plus, One Color” protocol (Agilent using gene expression microarrays. We performed degra- Technologies, Inc. 2007; Cat. N°: 5188-5339) and the dation by heat-treatment of isolated RNA to avoid tran- Agilent RNA Spike-In Kit for One color (Agilent Tech- scriptional processes in the context of cell death. nologies, Inc. 2007; Cat. N°: 5188-5282) following the manufacturer’s standard protocol. Global gene expres- Methods sion analysis was applied using the Human 4 × 44 K Samples and sample preparation design array from Agilent Technologies (G4112F). 600 Tumor biopsies from three patients with locally advanced ng of total RNA were used as a