Impact of Thawing on RNA Integrity and Gene Expression Analysis in Fresh Frozen Tissue : Diagnostic Molecular Pathology

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00019606-200903000-00006ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2009 Lippincott Williams & Wilkins, Inc.18March 2009 p 44-52Impact of Thawing on RNA Integrity and Gene Expression Analysis in Fresh Frozen TissueOriginal ArticlesBotling, 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*Departments of *Genetics and Pathology†Urology‡Otorhinolaryngology, University Hospital, Uppsala, SwedenThe 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: [email protected]).AbstractBiobanks 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.Clinical tissue biobanks provide a unique resource to study molecular changes in the in situ environment of human diseases.1 The acquisition of human tissues for extraction of the RNA and consequent gene expression studies is a critical challenge. RNA fragility and degradation are omnipresent concerns.As formalin-fixation often disqualifies tissue from RNA analysis, tremendous efforts have been made to identify optimal tissue procurement procedures for biobanking of unfixed tissue.2–5 We and others have shown that as long as the cellular structure is not disrupted, RNA integrity and gene expression levels are not significantly influenced in fresh, unfixed tissue, even at room temperature for several hours.6–9 Currently, transportation on ice, snap freezing and storage in low-temperature freezers are regarded as gold standards for most biobanking projects and provide sufficient tissue quality for molecular analysis.A number of studies have evaluated prefreezing conditions,6,10–13 but only a few studies have addressed the impact of tissue handling in the poststorage phase. After removal of tissue from the low-temperature freezer, the process of thawing destroys the cellular structure and degradation activities are initiated that may ultimately influence experimental results. With regard to RNA, the current belief is that degradation occurs immediately at room temperature and that thawing therefore should be avoided at all circumstances.14 However, in practice, this is not always possible. Already when frozen tissue specimens are sectioned in a cryostat, the transfer and attachment to the glass slide includes a short warm-up period. Additionally, repetitive thawing and freezing may occur when the same sample is used for a variety of analyses over time in different research projects. Obviously, these variables may lead to undesirable molecular degradation and alterations in valuable ex vivo material.The aim of our study was to evaluate the kinetics of RNA degradation after thawing of fresh frozen clinical tissue samples and the effect of RNA stabilization by RNAlater during thawing. Also, the impact of thawing-induced RNA degradation on gene expression was investigated. Additionally, the influence of repetitive thawing and freezing was examined. Finally, an acceptable grade of RNA degradation that does not significantly influence specific mRNA levels was determined.MATERIALS AND METHODSTissue Samples and Study DesignFully anonymized human tissue was used in agreement with the Swedish Biobank Legislation and in accordance with the ethical rules of the Department of Pathology, Uppsala University Hospital. Fresh tonsil tissue was obtained immediately after surgery and was cut in cores, approximately 1 mm in diameter and 10 mm long. These core biopsies were either transferred to empty cryotubes or cryotubes containing 0.5 mL of RNAlater solution (Ambion, Foster City) and snap frozen in isopentane/dry ice (−120°C) before storage in a low-temperature freezer at −80°C. After 1 week, the samples were removed from the freezer and directly transferred to the extraction buffer (time point 0 min) or thawed at room temperature for 5 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, and 16 hours, respectively, before RNA extraction. The study design is illustrated in Figure 1. RNA preparations from time points 0 minute, 5 minutes, 30 minutes, 45 minutes, 3 hours, and 16 hours were used for subsequent real-time polymerase chain reaction (RT-PCR) analyses. For the repetitive thawing and freezing experiment, similar fresh tonsil cores and cores suspended in RNAlater were used. The samples were repeatedly thawed for 5 minutes at room temperature and snap frozen in isopentane/dry ice for 10 minutes (once, 3 times, and 6 times, respectively; Fig. 3).JOURNAL/dimp/04.03/00019606-200903000-00006/figure1-6/v/2021-02-17T200000Z/r/image-jpeg Experimental design: fresh surgical tonsil specimens were cut in cores of equal size (approximately 10×1 mm). The core biopsies were transferred either into empty cryotubes (fresh frozen) or into cryotubes containing RNAlater and snap-frozen in isopentane/dry ice before being stored in a −80°C freezer for 1 week. The samples were removed from the freezer and thawed at room temperature for 0 minute, 5 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, and 16 hours, respectively, before RNA extraction. After RNA purification, the amount and quality of RNA was analyzed using microchip gel electrophoresis. RNA preparations from time points 0 minute, 5 minutes, 30 minutes, 45 minutes, 3 hours, and 16 hours were used for subsequent real-time polymerase chain reaction analysis.RNA Extraction and Microchip Gel ElectrophoresisTotal RNA was isolated from the tissue samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The quality and concentration of the RNA was assessed using the Agilent 2100 Bioanalyzer and RNA 6000 Nano Labchip Kit (Agilent Biotechnologies, Palo Alto). RNA sample (1 μL) was transferred to the NanoLabchip, together with 1 μL of RNA 6000 ladder (Ambion). The analysis was performed according to the manufacturer's instructions. Results are presented as electropherograms, and RNA Integrity Number (RIN) values were calculated with the provided software (www.agilent.com/chem/RIN). The RIN value is based on an algorithm that takes different aspects of the electropherogram curve into account, that is, shape and ratio of the 28S and 18S ribosomal RNA peaks, baseline configuration and presence of aberrant peaks composed of small degraded RNA fragments. A quality value is thus assigned to the sample, ranging from 10 (perfect quality) to 0 (totally degraded).15cDNA Synthesis and Quantitative RT-PCRcDNA was transcribed from 1 μg of total RNA, adding 1 μL oligo(dT) primers (500 μg/mL; Invitrogen, Carlsbad) and 1 μL of 10 mM dNTP Mix (Advantage UltraPure, Clontech, Mountain View). The mixture was heated to 65°C for 10 minutes and then immediately chilled on ice. To yield a total volume of 20 μL, 4 μL of 5× First-Strand Buffer, 2 μL of 0.1 M dithiothreitol, 1 μL of RNAsin (Promega, Madison), and 1 μL of SuperScript II reverse transcriptase (200 U) (Invitrogen) were added, and the reaction mixture was incubated at 42°C for 1 hour. After enzyme inactivation at 65°C for 20 minutes, the RNA was digested with RNaseH (Clontech) for 30 minutes at 37°C. cDNA (2 μL) was used for a 40 cycle quantitative RT-PCR assay using the SYBRgreen PCR Master Mix (Applied Biosystems, Foster City) with the primers described in Table 1. The reaction was performed on the ABI PRISM 7000HT RT-PCR cycler (Applied Biosystems) under conditions recommended by the manufacturer. Expression levels were calculated from experimental quadruplicates of 2 independent RNA preparations using the ΔΔCT method. The threshold cycle numbers (CT values) of the test genes were corrected to the CT values of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or hypoxanthine phosphoribosyl-transferase 1 (HPRT1). The normalized expression levels (ΔCTtest) of the genes were compared with the expression levels (ΔCTcontrol) of the reference samples (time point 0 minute, set to 1.0 for relative comparison).JOURNAL/dimp/04.03/00019606-200903000-00006/table1-6/v/2021-02-17T200000Z/r/image-tiff Primer Sequences and Concentrations Used for Real-time PCRRESULTSKinetics of RNA Degradation in Fresh Frozen Tissue Samples After ThawingTo evaluate the influence of thawing time under controlled conditions, fresh tonsil tissue, untreated or suspended in RNAlater, was used as described in Figure 1. Tubes containing tissue cores, with and without RNAlater, were removed from the freezer, thawed and left at room temperature in duplicates for 0 minute (reference sample), 5 minutes, 30 minutes, 45 minutes, 3 hours, 6 hours, and 16 hours before RNA extraction. The RNA quality was assessed on the basis of electropherogram images of microchip gel electrophoresis and by the RIN (Fig. 2). In one of the untreated duplicate tonsil samples, minimal signs of degradation were seen 30 minutes after thawing at room temperature reflected by a slight reduction of RIN values (7.1 and 9.3). Degradation of RNA was more pronounced at 45 minutes (RIN values 7.1 and 7.4) with a substantial elevation of the baseline between the two ribosomal peaks. After 6 hours at room temperature, distinct 18S and 28S peaks were still visible, indicating the presence of remaining long RNA fragments. Not until after 16 hours did the ribosomal peaks disappear completely due to advanced degradation. In the samples that were stored and thawed in RNAlater, no signs of degradation could be detected, even after prolonged thawing time (median RIN value 9.4 over the whole series of thawed samples). One outlier was detected at 6 hours (RIN 6.7).JOURNAL/dimp/04.03/00019606-200903000-00006/figure2-6/v/2021-02-17T200000Z/r/image-jpeg RNA integrity of tonsil tissue after thawing. Purified RNA was analyzed using microchip gel electrophoresis (Bioanalyzer). Representative electropherograms (fresh or incubated in RNAlater) of RNA obtained from thawed tissue kept at room temperature for different time periods (0 min, 5 min, 30 min, 45 min, 1 h, 3 h, 6 h, and 16 h) and the corresponding RNA integrity numbers of the duplicates are given.RNA Integrity After Repetitive Freezing and ThawingTo evaluate the impact of repetitive thawing and freezing, untreated fresh tonsil tissue cores, and tissue cores suspended in RNAlater were frozen and thawed once, 3 times, and 6 times, respectively (Fig. 3). The thawing period was standardized to 5 minutes. Minimal degradation occurred after 6 times of freezing and thawing with slightly decreased RIN values (8.4 and 7.5). With regard to total thawing time at room temperature (6×5 min=30 min), the degradation pattern corresponded to samples that were kept at room temperature continuously for 30 minutes (compare Figs. 2 and 3). Thus, the repetitive thawing process itself had no additional detrimental effect on RNA fragment integrity. When the tissue was suspended in RNAlater, the RNA transcripts remained intact even after 6 freezing and thawing cycles.JOURNAL/dimp/04.03/00019606-200903000-00006/figure3-6/v/2021-02-17T200000Z/r/image-jpeg RNA integrity of tonsil tissue after repetitive thawing. Purified RNA was analyzed using microchip gel electrophoresis (Bioanalyzer). RNA was extracted from tonsil tissue cores (fresh frozen or in RNAlater) after repetitive freezing and 5 minutes thawing cycles (1×, 3×, 6×). Representative electropherograms and the corresponding RNA integrity numbers of the duplicates are given.Gene Expression Analysis in Tissue Samples After Degradation due to ThawingTo investigate how thawing of human tissue samples and subsequent RNA degradation may induce alterations in gene expression, the expression of 5 different genes were analyzed using quantitative RT-PCR. The genes were selected to represent markers of different cellular regulatory pathways: FOS (immediate early response gene), TGFB1 (growth factor), HIF1A (hypoxia response), BCL2 (apoptosis regulator), and PCNA (cell cycle). In the RNAlater incubated tissue, all marker genes remained relatively stable at most time points (Fig. 4A). Minor changes in expression were only seen for FOS at 5 minutes and PCNA at 3 hours. These changes most likely reflect an expected variance between the different tonsil fractions analyzed at the different time points. In contrast, relevant alterations in gene expression were detected in 3 of 5 analyzed genes in the samples kept at room temperature for 45 minutes (Fig. 4B). A relevant increase (approximately 10-fold) in the detected expression of FOS and BCL2 correlated with the occurrence of minor degradation (RIN values: 7.1 and 7.4). In parallel, an increase in the threshold cycle number (CT value) of the reference housekeeping gene GAPDH was seen. For instance, in the analysis of FOS, the CT values of GAPDH (0 h: 24.2±0.5; 5 min: 24.4±0.6; 30 min 28.7±0.7; 45 min 30.1±0.9; 3 h 31.3±0.6; and 16 h 31.6±0.4) increased more than the CT values of FOS (0h: 28.4±0.6; 5 min: 29.8±1.8; 30 min 33.5±0.9; 45 min 31.0±1.2; 3 h 30.8±0.3; and 16 h 33.6±0.5), resulting in a higher relative expression level of FOS. A similar pattern was observed when BCL2 was analyzed. Conversely, along with prolonged thawing time, the CT values of TGFB1 increased even more rapidly than the CT values of GAPDH resulting in decreased normalized expression values for TGFB1 (Fig. 4B). HIF1A and PCNA exhibited an increase in CT values that paralleled the housekeeping gene and consequently the expression values did not change dramatically along with deteriorating RNA quality (Fig. 4B).JOURNAL/dimp/04.03/00019606-200903000-00006/figure4-6/v/2021-02-17T200000Z/r/image-jpeg Gene expression levels after thawing. RNA isolated from tonsil tissue that was kept at room temperature after thawing in RNAlater (A) or unfixed (B) was transcribed to cDNA using oligo-dT primers and analyzed using primers for FOS, HIF1A, PCNA, BCL2, and TGFB1. The results were normalized to GAPDH and given as mean±SD. Relative gene expression after different time periods (5 min, 30 min, 45 min, 3 h, and 16 h) was compared with time point 0 minute.As primer design may accidentally bias for certain transcripts, two new reference gene primer sets were designed: first one for an alternative housekeeping gene HPRT1, and a second one representing a shorter amplicon located closer to the 3′-end of the GAPDH transcript (GAPDH2) (Table 1). The results are shown in Figure 5. The amplitude of the relative increase for FOS expression was in general lower when GAPDH2 and HPRT1 primer sets were used for normalization. However, the changes in relative gene expression after thawing for 30 and 45 minutes were still detectable. The expression changes for BCL2 were nearly normalized when the newly designed housekeeping gene primers were used (Fig. 5B).JOURNAL/dimp/04.03/00019606-200903000-00006/figure5-6/v/2021-02-17T200000Z/r/image-jpeg Gene expression after thawing using different housekeeping gene primers. RNA isolated from tonsil tissue after thawing was transcribed to cDNA using oligo-dT primers and analyzed using primers for FOS (A) and BCL2 (B). Two new primer sets were designed for the housekeeping gene GAPDH2 (middle columns) and HPRT1 (right columns). The relative gene expression based on the normalization against the new control genes (HPRT1, GAPDH2) were compared with the previous calculations (GAPDH1; left columns) and given as mean±SD.DISCUSSIONThe fragility of RNA is a general obstacle when human tissue is used for gene expression analysis. Since most studies evaluated the impact of prefreezing conditions on RNA integrity in human biobank samples, this study instead focused on the time course after thawing of unfixed tissue, and tissue immersed in RNAlater.After thawing, minimal degradation occurred not in seconds or few minutes but after approximately 30 minutes at room temperature. Repetitive thawing and freezing was also not detrimental to RNA as long as the total thawing time was short. Both observations are of practical relevance, as certain techniques demand short periods of thawing. This is particularly true for thawed tissue attached to a slide during laser microdissection or in-situ hybridization, or during transfer and immersion into fixatives.16–18 Furthermore, frozen tissue blocks that have been thawed, accidentally or during some experimental procedures, do not necessarily have to be disqualified from further use in gene expression analysis.However, the kinetics of RNA degradation after thawing is likely to be tissue specific. Still we believe our findings with regard to RNA degradation kinetics in tonsil tissue is representative for the majority of human solid tissues. However, in tissues with high RNase activity the time course of RNA breakdown may be faster.Once degradation occurs, even if it is only marginal, gene expression results have to be interpreted with extreme caution. The RNA degradation after 1 and 3 hours of thawing may be still regarded as moderate,19 with 2 clearly visible ribosomal peaks and a RIN value of 6.5. In contrast to the moderate changes in the Bioanalyzer electropherograms, the calculated expression levels for particular genes were tremendously altered and consequently must be regarded as not informative.When relative gene expression is measured by quantitative RT-PCR the used housekeeping genes should be carefully selected, and optimally several housekeeping genes should be included.20 In our study, the inclusion of a different housekeeping gene (HPRT1) was able to compensate for the differences in gene expression observed for BCL2. However, for FOS this strategy was insufficient to balance the degradation-induced alterations. The changes of the CT values suggested that the housekeeping genes GAPDH and HPRT1 are more fragile to degradation compared with the FOS transcripts. In contrast, the TGFB1 transcripts seemed to be even more susceptible to degradation resulting in decreased normalized expression values in the thawed samples after 30 minutes. Finally, HIF1A and PCNA showed a degradation pattern comparable with that of GAPDH reflected by a similar CT increase and consequently stable normalized expression values.Thus, degradation after thawing in unfixed tissue is variable and transcript dependent. Therefore, it is difficult to predict if measured values reflect true expression, if they are due to transcript-specific degradation or the result of primer design. In this respect, it is important to stress that in all samples and all tested genes variable expression levels were solely seen in samples with decreased RIN values, indicating that the RIN value is a very suitable marker for the performance of quantitative RT-PCR. In our study, the critical RIN value for this process, associated with potentially erroneous expression values, was below 8. Optimally, expression analysis should only be performed after quality check and only high-quality RNA should be used.RNA degradation may occur under numerous conditions: physiologic RNA decay, RNA breakdown induced by necrosis, apoptosis, or ischemia in vivo, autolysis after tissue devitalization, RNA fragmentation during storage in freezers, thawing and handling of tissue, and in vitro degradation after RNA isolation.10,21,22 To our knowledge, this study is the first to systematically examine the effects of thawing in clinically relevant human tissue specimens in a controlled, experimental setting. The observation that RNA degradation is crucial for exact gene expression quantification is in accordance with a recent study analyzing mRNA expression of 3 housekeeping genes in commercially available reference RNA of different quality metrics.15 Another study used RNA from bovine tissue that was degraded enzymatically or under ultraviolet light. A strong dependency of the absolute quantification represented by the threshold values of the amplification curves (CT values) was observed.23 Antonov and coworkers24 describe the relation between RNA integrity, amplicon size, and CT values in a homogenous system using hydrolyzed RNA from a cell line. The degradation-induced alterations could be partially compensated by calculating ΔCT values between test genes and the mean values of several control genes.24 These studies and our results were based on RT-PCR analysis and did not include array-based methods. A few studies have examined the impact of degradation on global expression analysis using microarrays. In a systematic study, RNA that was degraded to different extent by endogenous RNases was linearly amplified and analyzed on an Affymetrix microarray platform. The number of identified regulated genes decreased with the use of moderately degraded RNA. But the authors conclude that the sensitivity was only slightly affected and meaningful results still might be obtained with moderately degraded samples.19 However, most researchers agree that also for global expression analysis using microarrays strict RNA quality criteria should be applied.13,22,25,26Our study revealed that RNAlater prevents RNA degradation in tissue and stabilizes the expression levels of selected genes. This result corresponds to previous studies that tested the use of RNAlater in biobank protocols. Florell et al4 showed that RNA of excellent quality and reliable expression patterns could be obtained even after 2 weeks tissue storage at 4°C. Mutter et al27 confirmed that RNAlater did not significantly influence RNA expression on Affymetrix arrays. Nevertheless, some studies indicate disadvantages in terms of tissue morphology when RNAlater is used and that snap freezing in liquid nitrogen provide comparable or even better RNA quality.3,16 Therefore, we still regard snap frozen tissue as the “gold standard” for RNA analysis when adequate quality tests are included.In conclusion, our study indicates that minimal RNA degradation occurs first after 30 minutes. This variable and transcript dependent degradation makes it very difficult to predict if the measured RNA values are truly specific and representative of gene expression. Incubation in RNAlater provides an efficient protection against this degradation. Finally, to avoid the introduction of artificial expression changes, the bioanalyzer-based RIN values provide good-quality metrics and only RNA preparations with high RIN values should be used in quantitative gene expression analysis. Consequently, thorough RNA integrity assessment is recommended before all RNA analysis techniques using fresh frozen tissue specimens.ACKNOWLEDGMENTThe authors thank Dr Mitsuhiro Ohshima (Department of Biochemistry, School of Dentistry, Nihon University, Tokyo, Japan) for critically reviewing this manuscript and for his helpful advice.REFERENCES1. Crawford JM, Tykocinski ML. Pathology as the enabler of human research. Lab Invest. 2005;85:1058–1064.[Context Link][CrossRef][Medline Link]2. Wester K, Asplund A, Backvall H, et al. Zinc-based fixative improves preservation of genomic DNA and proteins in histoprocessing of human tissues. Lab Invest. 2003;83:889–899.[Context Link][CrossRef][Medline Link]3. Wang SS, Sherman ME, Rader JS, et al. Cervical tissue collection methods for RNA preservation: comparison of snap-frozen, ethanol-fixed, and RNAlater fixation. Diagn Mol Pathol. 2006;15:144–148.[Context Link][Full Text][CrossRef][Medline Link]4. Florell SR, Coffin CM, Holden JA, et al. 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The core biopsies were transferred either into empty cryotubes (fresh frozen) or into cryotubes containing RNAlater and snap-frozen in isopentane/dry ice before being stored in a −80°C freezer for 1 week. The samples were removed from the freezer and thawed at room temperature for 0 minute, 5 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, and 16 hours, respectively, before RNA extraction. After RNA purification, the amount and quality of RNA was analyzed using microchip gel electrophoresis. RNA preparations from time points 0 minute, 5 minutes, 30 minutes, 45 minutes, 3 hours, and 16 hours were used for subsequent real-time polymerase chain reaction analysis. Primer Sequences and Concentrations Used for Real-time PCR RNA integrity of tonsil tissue after thawing. Purified RNA was analyzed using microchip gel electrophoresis (Bioanalyzer). Representative electropherograms (fresh or incubated in RNAlater) of RNA obtained from thawed tissue kept at room temperature for different time periods (0 min, 5 min, 30 min, 45 min, 1 h, 3 h, 6 h, and 16 h) and the corresponding RNA integrity numbers of the duplicates are given. RNA integrity of tonsil tissue after repetitive thawing. Purified RNA was analyzed using microchip gel electrophoresis (Bioanalyzer). RNA was extracted from tonsil tissue cores (fresh frozen or in RNAlater) after repetitive freezing and 5 minutes thawing cycles (1×, 3×, 6×). Representative electropherograms and the corresponding RNA integrity numbers of the duplicates are given. Gene expression levels after thawing. RNA isolated from tonsil tissue that was kept at room temperature after thawing in RNAlater (A) or unfixed (B) was transcribed to cDNA using oligo-dT primers and analyzed using primers for FOS, HIF1A, PCNA, BCL2, and TGFB1. The results were normalized to GAPDH and given as mean±SD. Relative gene expression after different time periods (5 min, 30 min, 45 min, 3 h, and 16 h) was compared with time point 0 minute. Gene expression after thawing using different housekeeping gene primers. RNA isolated from tonsil tissue after thawing was transcribed to cDNA using oligo-dT primers and analyzed using primers for FOS (A) and BCL2 (B). Two new primer sets were designed for the housekeeping gene GAPDH2 (middle columns) and HPRT1 (right columns). The relative gene expression based on the normalization against the new control genes (HPRT1, GAPDH2) were compared with the previous calculations (GAPDH1; left columns) and given as mean±SD.Impact of Thawing on RNA Integrity and Gene Expression Analysis in Fresh Frozen TissueBotling 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 MDOriginal ArticlesOriginal Articles118p 44-52