Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of gene expression profiling of mouse tissues obtained during the postmortem interval

ABSTRACT: Attempts to establish a tissue bank from autopsy samples have led to uncovering of the secrets of many diseases. Here, we examined the length of time that the RNA from postmortem tissues is available for microarray analysis and reported the gene expression profile for up- and down-regulated genes during the postmortem interval (PMI). We extracted RNA from fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) brains and livers of three different groups of mice: 1) mice immediately after death, 2) mice that were stored at room temperature for 3 h after death, and 3) mice that were stored at 4Â°C for 18 h after death, as this storage resembles the human autopsy process in Japan. Based on the microarray analysis, we selected genes that were altered by >1.3-fold or <0.77-fold and classified these genes using hierarchical cluster analysis following DAVID (database for annotation, visualization, and integrated discovery) gene ontology analysis. These studies revealed that cytoskeleton-related genes were enriched in the set of up-regulated genes, while serine protease inhibitors were enriched in the set of down-regulated genes. Interestingly, although the RNA quality was maintained, up-regulated genes were not validated by quantitative PCR, suggesting that these genes may become fragmented or modified by an unknown mechanism. We extracted RNA from fresh-frozen (FF) brains and livers from mice under three different conditions: 1) mice just after death as a control, 2) mice that were stored at room temperature for 3 h after death, and 3) mice that were stored at 4Â°C for 18 h after death to resemble the human autopsy process. We also created formalin-fixed paraffin-embedded (FFPE) tissue blocks at the same time using mouse organs obtained under the three conditions described above. We then purified RNA from the FFPE tissue blocks. Furthermore, we performed microarray analysis to examine changes in the gene expression profiles during the postmortem interval in FF samples and to compare ene expression profiles between FF and FFPE samples at three different postmortem times as described for the FF samples.

ORGANISM(S): Mus musculus

SUBMITTER: Masatoshi Ichihara

PROVIDER: E-GEOD-78203 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This study was performed to evaluated RNA extraction and gene expression analysis of FFPE specimen stored for more than 20 years. Using long time stored FFPE material; large retrospective studies correlating molecular features with therapeutic response and clinical outcome, can be performed. Quantitative PCR (qPCR) was used to evaluate RNA extraction methods and to compare gene expression profiles of FFPE and fresh frozen (FF) tissue. Extracted RNA was subsequently subjected to microarray analysis and compared to qPCR data. The Ambion RecoverAll kit appears to be particularly suited for RNA extraction of long time stored FFPE tissues. Gene expression analysis using Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues. We conclude that high quality gene expression signatures can be recognized using Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality. Three RNA extraction methods from Roche (Basel, Switzerland), Ambion (Austin, TX, USA) and Qiagen (Hilden, Germany), designed for FFPE material was used. The methods were compared using qPCR. For the qPCR analysis, two different concentrations of input cDNA were used (20ng and 100ng) and 32 human endogenous control genes were examined. RNA from the Ambion FFPE kit was further analyzed by using microarray. Amplification of RNA prior to microarray analysis was performed using Nugen technologies (San Carlos, CA, USA). Nugen has developed amplification kits both for FFPE and FF materials: WT- ovation FFPE RNA amplification System V2 (Nugen FFPE), Ovation FF RNA Amplification System V2 (Nugen V2 FF) and Ovation FF Pico RNA Amplification System (Nugen PICO FF). The Nugen FFPE kit, designed for FFPE material was only used for FFPE tissue. The Nugen Pico FF kit, designed to target small amounts of FF RNA (>500 pg) and the Nugen V2 FF kit designed to target total FF RNA, were only used for FF tissue. Affymetrix standard amplification protocol (Affy FF) designed for FF RNA was also included as the standard method for amplification.

Project description:As proteins represent cell function, proteomic analysis of prostate tissues may elucidate the mechanisms of prostate cancer (PCa) progression. To that end, formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources, but associated with large analytical difficulties due to protein cross-linking. Our aim is to characterize tissue protein changes associated with castration resistant PCa. This study focused at the development of a reliable protocol for the proteomic analysis of FFPE tissues which was followed by a pilot study using FFPE PCa clinical samples to investigate whether the optimized protocol can provide biologically relevant data in the context of PCa. Archival FFPE mouse tissues were processed using seven protein extraction protocols including combinations of homogenization means (beads, sonication, boiling) and buffers (Tris-HCl and Urea-Thiourea). The proteomics output was evaluated by SDS electrophoresis and high resolution LC-MS/MS analysis. FFPE tissues (3 sections, 15��m each per sample) from 10 patients with PCa corresponding to tumor (GS=6 or GS��8) and adjacent benign regions were processed with the optimized protocol. Extracted proteins were analyzed by GeLC-MS/MS followed by statistical and bioinformatics analysis using Cytsoscape and Open Targets tool. A combination of cell lysis by Tris-HCl, SDS and DTE with bead homogenization, sonication and boiling provided most efficient protein extraction from FFPE tissues based on protein identifications and reproducibility. Comparison between the FFPE and matched FF tissues showed a substantial overlap in protein identifications (502 common out of 1,216 IDs commonly identified in all FF samples and 530 IDs commonly identified in all FFPE samples) with a strong correlation in relative abundances (rs=0.846, p<.001). Proteins significantly deregulated between PCa GS��8 and PCa GS=6 represented extracellular matrix organisation, phosphorylation and gluconeogenesis pathways, while proteins deregulated between cancerous tissues and benign counterparts, reflected increased translation, peptide synthesis and protein metabolism in the former, as expected. Further disease association analysis using the Open Targets platform showed that 60% of the differentially expressed proteins in cancer versus normal adjacent were significantly associated with PCa. These results support the relevance of the proteomic findings in the context of PCa and the reliability of the optimized protocol for proteomics analysis of FFPE material. A large scale study including 160 clinical FFPE prostate cancer and benign tissues to define protein changes associated with aggressive PCa, is planned.

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Characterization of gene expression profiling of mouse tissues obtained during the postmortem interval

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