Effects of Ischemia on Gene Expression (original) (raw)
Molecular Oncology, 2014
Background: Tissue handling can alter global gene expression potentially affecting the analytical performance of genomic signatures, but such effects have not been systematically evaluated. Methods: Tissue samples from 11 previously untreated breast tumors were minced and aliquots were either snap frozen or placed in RNAlater immediately or after 20, 40, 60, 120 or 180 min at room temperature. RNA was profiled on Affymetrix HG-U133A arrays. We used probe-set-wise hierarchical models to evaluate the effect of preservation method on transcript expression and linear mixed effects models to assess the effect of cold ischemic delay on the expression of individual probe sets. Gene set enrichment analysis identified pathways overrepresented in the affected transcripts. We combined the levels of 41 most sensitive transcripts to develop an index of ischemic stress. Results: Concordance in global gene expression between the baseline and 40 min delay was higher for samples preserved in RNAlater (average concordance correlation coefficient CCC ¼ 0.92 compared to 0.88 for snap frozen). Overall, 481 transcripts (3%) were significantly affected by the preservation method, most of them involved in processes important in cancer. Prolonged cold ischemic delay of up to 3 h induced marginal global gene expression changes (average CCC ¼ 0.90 between baseline and 3 h delay). However 41 transcripts were significantly affected by cold ischemic delay. Among the induced transcripts were stress response genes, apoptotic response genes; among the downregulated were genes involved in metabolism, protein processing and cell cycle regulation. An index combining the expression levels of these genes was proportional to the cold ischemic delay. Conclusions: Prolonged cold ischemia induces significant transcriptional changes in a small subset of transcripts in the tissue. Furthermore, the expression level of about 3% of the transcripts is affected by the preservation method. These sensitive transcripts should not be included in genomic signatures for more reliable analytical performance.
PLoS ONE, 2013
Background: Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures. Methods: RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method. Results: Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation. Conclusions: Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.
Analytical Biochemistry, 2004
Factors such as warm ischemia and time at room temperature before tissue treatment may inXuence the results of mRNA expression analyses on tissue specimens obtained during surgery. We evaluated the eVect of these factors on RNA integrity and mRNA expression levels by incubating freshly obtained mouse liver tissue at 25 or 37°C for periods of 0-4 h. Changes in the mRNA expression levels of seven genes, Tbp, Eef1a, Fos, Junb, Myc, Vegf, and Glut2, were determined by real-time reverse transcription-polymerase chain reaction. Incubation at 25°C for up to 4 h only slightly altered (by a factor of less than 2) levels of mRNA for Tbp, Eef1a, Junb, Myc, Vegf, and Glut2. This result is consistent with limited RNA degradation at this temperature. Incubation at 37°C strongly aVected the levels of these mRNAs. Four hours of incubation at this temperature resulted in extensive RNA degradation, with mRNA levels falling to 1/10th those before incubation. When relative quantiWcation was performed, i.e., quantiWcation of the target gene transcripts in comparison to an endogenous housekeeping transcript (Tbp or Eef1a), the changes in mRNA levels were reduced to less than 2.5-fold. Fos behaved very diVerently from the other genes tested on incubation, with Fos mRNA levels increasing considerably following incubation at either 25 or 37°C. Our data suggest that, with the exception of certain genes induced by tissue injury, relative quantiWcation of mRNA, even on degraded RNA samples, can provide a reliable estimate of in vivo mRNA levels.
2004
Factors such as warm ischemia and time at room temperature before tissue treatment may inXuence the results of mRNA expression analyses on tissue specimens obtained during surgery. We evaluated the eVect of these factors on RNA integrity and mRNA expression levels by incubating freshly obtained mouse liver tissue at 25 or 37°C for periods of 0-4 h. Changes in the mRNA expression levels of seven genes, Tbp, Eef1a, Fos, Junb, Myc, Vegf, and Glut2, were determined by real-time reverse transcription-polymerase chain reaction. Incubation at 25°C for up to 4 h only slightly altered (by a factor of less than 2) levels of mRNA for Tbp, Eef1a, Junb, Myc, Vegf, and Glut2. This result is consistent with limited RNA degradation at this temperature. Incubation at 37°C strongly aVected the levels of these mRNAs. Four hours of incubation at this temperature resulted in extensive RNA degradation, with mRNA levels falling to 1/10th those before incubation. When relative quantiWcation was performed, i.e., quantiWcation of the target gene transcripts in comparison to an endogenous housekeeping transcript (Tbp or Eef1a), the changes in mRNA levels were reduced to less than 2.5-fold. Fos behaved very diVerently from the other genes tested on incubation, with Fos mRNA levels increasing considerably following incubation at either 25 or 37°C. Our data suggest that, with the exception of certain genes induced by tissue injury, relative quantiWcation of mRNA, even on degraded RNA samples, can provide a reliable estimate of in vivo mRNA levels.
The effects of death and post-mortem cold ischemia on human tissue transcriptomes
Nature communications, 2018
Post-mortem tissues samples are a key resource for investigating patterns of gene expression. However, the processes triggered by death and the post-mortem interval (PMI) can significantly alter physiologically normal RNA levels. We investigate the impact of PMI on gene expression using data from multiple tissues of post-mortem donors obtained from the GTEx project. We find that many genes change expression over relatively short PMIs in a tissue-specific manner, but this potentially confounding effect in a biological analysis can be minimized by taking into account appropriate covariates. By comparing ante- and post-mortem blood samples, we identify the cascade of transcriptional events triggered by death of the organism. These events do not appear to simply reflect stochastic variation resulting from mRNA degradation, but active and ongoing regulation of transcription. Finally, we develop a model to predict the time since death from the analysis of the transcriptome of a few readil...
Effects of RNA degradation on gene expression analysis of human postmortem tissues
Faseb Journal, 2005
The Affymetrix GeneChip platform was used to build a gene expression database of the normal human body. Postmortem human tissues represent a valuable source of biological materials for this type of study, but their use entails some delays before harvesting such tissues. We first evaluated the RNA quality obtained from tissues obtained 3-5 h postmortem and found variations that were both tissue and donor-dependent. RNAs extracted from brain regions were of higher quality than those obtained from the gut, while the cause of death was a significant factor in donor-dependent differences. To avoid these variables, we used rat duodenum to determine the effects of RNA degradation on the analysis of gene expression. Surprisingly, even samples exhibiting significant RNA degradation yielded robust gene expression results, comparable to those obtained using intact samples at a certain signal intensity cutoff. We extended these findings to our human expression database and obtained similar results, indicating that the Affymetrix platform, which is biased to the 3′ end of transcripts for detection, can tolerate significant RNA degradation, while still yielding high quality expression data. Our resulting body index expression database is a valuable research tool. As examples of potential uses, we report novel expression sites for four potential therapeutic targets-CCL27, GPR22, GPR113 and GPR128-and as well as a set of thymus-specific genes, including three not previously associated with the thymus.
Translation-State Analysis of Gene Expression in Mouse Brain After Focal Ischemia
Journal of Cerebral Blood Flow & Metabolism, 2004
Confounding any genome-scale analysis of gene expression after cerebral ischemia is massive suppression of protein synthesis. This inefficient translation questions the utility of examining profiles of total transcripts. Our approach to such postischemic gene profiling in the mouse by microarray analysis was to concentrate on those mRNAs bound to polyribosomes. In our proof-of-principle study, polysomally bound and unbound mRNAs were subjected to microarray analysis: of the 1,161 transcripts that we found to increase after ischemia, only 36% were bound to polyribosomes. In addition to the expected increases in heat-shock proteins and metallothioneins, increases in several other bound transcripts involved in the promotion of cell survival or antiinflammatory behavior were noted, such as CD63 (Lamp3), Lcn2 (lipocalin-2), Msn (moesin), and UCP2 (uncoupling protein 2), all of which showed
Biological Psychiatry, 2004
There are major concerns that specific agonal conditions, including coma and hypoxia, might affect ribonucleic acid (RNA) integrity in postmortem brain studies. We report that agonal factors significantly affect RNA integrity and have a major impact on gene expression profiles in microarrays. In contrast to agonal factors, gender, age, and postmortem factors have less effect on gene expression profiles. The Average Correlation Index is proposed as a method for evaluating RNA integrity on the basis of similarity of microarray profiles. Reducing the variance due to agonal factors is critical in investigating small but validated gene expression differences in messenger RNA levels between psychiatric patients and control subjects.