Unknown,Transcriptomics,Genomics,Proteomics

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MiRNA profiling from freshly harvested livers

ABSTRACT: Correlation of miRNA expression patterns with clinically relevant information is greatly facilitated by the retrospective analysis of samples archived in tissue banks. Unfortunately, the quality of samples stored in tissue banks is variable due to heterogeneity in pre-analytical preparation of clinical specimens. Collectively, these variables will impact the reliability of the results of the analysis. To date no systematic studies have been performed to investigate the relationship between total RNA degradation and miRNA profiles determined from snap-frozen collections or freshly harvested tissues. To investigate this question, we compared miRNA expression profiles generated through delaying the extraction of RNA from Liver and Duodenum, to generate different RNA integrities as defined by RIN values. Liver samples were collected from mice, sliced into five identical pieces, transfered into eppendorf tubes and either processed immediately (T0) or maintained on ice and at latter time points [30 min (T30), 60 min (T60), 120 min (T120) and 240 min (T240)]. RNA was extracted by using Trizol and RNA integrity was assessed by Bioanalyzer electropherograms. We found that liver samples did not show any significant RNA degradation even when samples remained on ice for up to 4 hrs However, miRNA expression profiling by microarray shows that miRNAs from freshly harvested Liver are partially degraded. Based on these data, we conclude that samples with low RIN values (less than 7) do not merit analysis on miRNA arrays.

ORGANISM(S): Mus musculus

SUBMITTER: Martina Muckenthaler

PROVIDER: E-GEOD-18775 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

RNA degradation compromises the reliability of microRNA expression profiling.

Ibberson David D Benes Vladimir V Muckenthaler Martina U MU Castoldi Mirco M

BMC biotechnology 20091221

<h4>Background</h4>MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally in ...[more]

PMID: 20025722

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Project description:To determine the effect of RNA degradation on miRNA expression profiling. To mimic total RNA degradation in snap-frozen livers and duodenums from mice (wild type female, C57/Bl6) tissues were stored in a -80Â°C freezer and then transferred to dry ice. Each frozen tissue was sliced into five identical pieces which then were transferred into eppendorf tubes. At time point zero (T0), all samples were placed on ice and total RNA was extracted immediately from (T0) and at later time points [30 min (T30), 60 min (T60), 120 min (T120) and 240 min (T240)]. RNA integrities were assessed using the Agilent Bioanalyzer 2100 which calculates RIN values of assayed RNAs. We found that at T0, the RNA integrity for both liver and duodenum was above 7, indicating good quality total RNA. However, 30 min on ice was sufficient to reduce RNA integrity, indicated by the decrease in RINs. These findings indicate that RNA degradation can take place in defrosted tissue at low temperature (i.e. on ice). For comparison, we assessed the extent to which RNA integrity is preserved in freshly harvested tissues when tissue processing is delayed. Liver and duodenum samples were collected from mice and either processed immediately or maintained on ice, as described above. Bioanalyzer electropherograms of the liver samples did not indicate any significant total RNA degradation even when samples remained on ice for up to 4 hrs. By contrast, duodenal samples (which are rich in RNases) do show high susceptibility to degradation similar to the snap-frozen defrosted material. These findings suggest that tissues such as duodenum or pancreas should either be processed immediately or snap-frozen and processed individually. To assess to which extent total RNA degradation affects miRNA expression profiles, we hybridized to the miCHIP microarray total RNAs extracted from freshly harvested livers and duodenums liver and from snap frozen livers and duodenums. Hierarchical clustering (HCL), by Pearson correlation, was used to cluster samples based on their miRNA expression profiles. Our analysis shows that miRNAs extracted from freshly harvested liver is less degraded (matrix plot analyses). Consistent with high level of RNases present in duodenum, miRNAs from freshly harvested duodenum are extensively degraded. Based on these data, we conclude that samples with low RIN values (less than 7) do not merit analysis on miRNA arrays.

Project description:Phagocytosis requires the activation of a plethora of mechanisms that include the activation of the actin cytoskeleton guided by the Arp2/3 complex. These are promoted by activators such as the Wiskott Aldrich Syndrome Protein (WASP) family members. In order to further understand the molecular mechanisms involved in the early events leading the phagocytosis of the pathogenic Mycobacterium tuberculosis, we set out to examine potential roles of miRNAs in phagocytosis using genome-wide expression profiling to identify miRNAs differentially regulated following mycobacterial infection. One of the miRNAs activated upon infection of mouse macrophages with the non-pathogenic Mycobacterium smegmatis, the widely conserved miR-142-3p, was predicted and confirmed to target the Neural-WASP (N-WASP). Upregulating of miR-142-3p in mouse macrophages inversely correlated with levels of N-WASP, upon infection with live pathogenic and non-pathogenic mycobacteria, suggesting an active role of Mycobacterium tuberculosis on the regulation of phagocytosis, at the post-transcriptional level, in host cells. The reduction of N-WASP correlated with a reduced internalization of bacteria per macrophage, independently of the phagocytosis index. Furthermore, the downregulation of WASP levels accompanied those of N-WASP, at early but not at late time points, suggesting a closely regulatory mechanism among both family members, dependent on the time frame of the phagocytosis. Additionally, upregulating of miR-142-3p promoted the change in the protein levels of another predicted and confirmed target, the Cofilin2 protein, in a phagocytosis-independent fashion. Downregulation experiments promoted aberrant morphologic phenotypes in macrophages, similar to observed by others in PBMCs of humans with Wiskott Aldrich Syndrome, suggesting the strong involvement of miR-142-3p on the regulation of the actin machinery in macrophages. Altogether these results show for the first time that miRNAs are involved in the regulation of actin-mediated phagocytosis of pathogenic bacteria and that these are direct targets of Mycobacterium tuberculosis. Expression analysis of mouse macrophage cell line J774A.1 in response to infection with Mycobacterium smegmatis mc2155 EGFP. Three biological replicates each for uninfected and infected samples.

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MiRNA profiling from freshly harvested livers

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RNA degradation compromises the reliability of microRNA expression profiling.

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