RNA degradation compromises the reliability of microRNA expression profiling

Background: 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 induced RNA degradation affects microRNA expression profiles. Results: Tissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP) or microRNA-specific real-time quantitative PCR (miQPCR). Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays. Conclusion: MicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven

MicroRNAs from saliva of anopheline mosquitoes mimic human endogenous miRNAs and may contribute to vector-host-pathogen interactions

During blood feeding haematophagous arthropods inject into their hosts a cocktail of salivary proteins whose main role is to counteract host haemostasis, inflammation and immunity. However, animal body fluids are known to also carry miRNAs. To get insights into saliva and salivary gland miRNA repertoires of the African malaria vector Anopheles coluzzii we used small RNA-Seq and identified 214 miRNAs, including tissue-enriched, sex-biased and putative novel anopheline miRNAs. Noteworthy, miRNAs were asymmetrically distributed between saliva and salivary glands, suggesting that selected miRNAs may be preferentially directed toward mosquito saliva. The evolutionary conservation of a subset of saliva miRNAs in Anopheles and Aedes mosquitoes, and in the tick Ixodes ricinus, supports the idea of a non-random occurrence pointing to their possible physiological role in blood feeding by arthropods. Strikingly, eleven of the most abundant An. coluzzi saliva miRNAs mimicked human miRNAs. Prediction analysis and search for experimentally validated targets indicated that miRNAs from An. coluzzii saliva may act on host mRNAs involved in immune and inflammatory responses. Overall, this study raises the intriguing hypothesis that miRNAs injected into vertebrates with vector saliva may contribute to host manipulation with possible implication for vector-host interaction and pathogen transmission

FLT-PET in previously untreated patients with low-grade glioma can predict their overall survival

BACKGROUND: Low-grade gliomas (LGG) of the brain have an uncertain prognosis, as many of them show continuous growth or upgrade over the course of time. We retrospectively investigated the role of positron emission tomography with 3’-deoxy-3’-[18F]fluorothymidine (FLT-PET) in the prediction of overall survival and event free survival in patients with untreated LGG. No such information is yet available in the literature. MATERIAL AND METHODS: Forty-one patients with previously untreated LGG underwent 55 FLT-PET investigations during their follow-up because of subjective complaints, objective worsening of clinical conditions, equivocal findings or progression on magnetic resonance imaging. The time interval before referral for neurosurgical or radiation treatment was considered to be event free survival, the interval until death as overall survival, respectively. Standardized uptake values (SUV) were measured, and a 3-point scale of subjective assessment was also applied. ROC analysis was used to define cut-off values. The log rank test was used for comparison of Kaplan-Meier survival curves. RESULTS: Eight patients (a total of 9 FLT-PET studies performed) died during follow-up. Progression leading to referral to therapy was recorded in 24 patients (a total of 33 FLT-PET studies). With a cut-off value of SUVmean = 0.236, a median overall survival of 1.007 days was observed in the test positive subgroup while median overall survival for the test negative subgroup was not achieved (p = 0.0002), hazard ratio = 17.6. Subjective assessment resulted in hazard ratio 11.5 (p = 0.0001). Only marginal significance (p=0.0562) was achieved in prediction of event free survival. CONCLUSIONS: Increased FLT uptake in previously untreated patients with LGG is a strong predictor of overall survival. On the other hand, prediction of event free survival was not successful in our cohort, probably because of high prevalence of patients who needed treatment due to symptoms caused by a space-occupying lesion without respect to the proliferative activity of the tumour

gga-miRNOME, a microRNA-sequencing dataset from chick embryonic tissues

MicroRNAs (miRNAs) are small non-coding RNA molecules, with sizes ranging from 18 to 25 nucleotides, which are key players in gene expression regulation. These molecules play an important role in fine-tuning early vertebrate embryo development. However, there are scarce publicly available miRNA datasets from non-mammal embryos, such as the chicken (Gallus gallus), which is a classical model system to study vertebrate embryogenesis. Here, we performed microRNA-sequencing to characterize the early stages of trunk and limb development in the chick embryo. For this, we profiled three chick embryonic tissues, namely, Undetermined Presomitic Mesoderm (PSM_U), Determined Presomitic Mesoderm (PSM_D) and Forelimb Distal Cyclic Domain (DCD). We identified 926 known miRNAs, and 1,141 novel candidate miRNAs, which nearly duplicates the number of Gallus gallus entries in the miRBase database. These data will greatly benefit the avian research community, particularly by highlighting new miRNAs potentially involved in the regulation of early vertebrate embryo development, that can be prioritized for further experimental testing.info:eu-repo/semantics/publishedVersio

VISOR: a versatile haplotype-aware structural variant simulator for short- and long-read sequencing.

Abstract Summary VISOR is a tool for haplotype-specific simulations of simple and complex structural variants (SVs). The method is applicable to haploid, diploid or higher ploidy simulations for bulk or single-cell sequencing data. SVs are implanted into FASTA haplotypes at single-basepair resolution, optionally with nearby single-nucleotide variants. Short or long reads are drawn at random from these haplotypes using standard error profiles. Double- or single-stranded data can be simulated and VISOR supports the generation of haplotype-tagged BAM files. The tool further includes methods to interactively visualize simulated variants in single-stranded data. The versatility of VISOR is unmet by comparable tools and it lays the foundation to simulate haplotype-resolved cancer heterogeneity data in bulk or at single-cell resolution. Availability and implementation VISOR is implemented in python 3.6, open-source and freely available at https://github.com/davidebolo1993/VISOR. Documentation is available at https://davidebolo1993.github.io/visordoc/. Supplementary information Supplementary data are available at Bioinformatics online

Identification of MiR-21-5p as a functional regulator of mesothelin expression using microRNA capture affinity coupled with next generation sequencing

MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3′untranslated region (3′UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs::mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA::MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-21-5p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA::mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth

Post flooding damage assessment of earth dams and historical reservoirs using non-invasive geophysical techniques

This paper describes the use of four geophysical techniques to map the structural integrity of historical earth reservoir embankments which are susceptible to natural decay with time. The four techniques that were used to assess the post flood damage were 1. A fast scanning technique using a dipole electromagnetic profile apparatus (GEM2), 2. Electrical Resistivity Tomography (ERT) in order to obtain a high resolution image of the shape of the damaged/seepage zone, 3. Self-Potential surveys were carried out to relate the detected seepage evolution and change of the water displacement inside the embankment, 4. The washed zone in the areas with piping was characterised with microgravimetry. The four geophysical techniques used were evaluated against the case studies of two reservoirs in South Bohemia, Czech Republic. A risk approach based on the Geophysical results was undertaken for the reservoir embankments. The four techniques together enabled a comprehensive non-invasive assessment whereby remedial action could be recommended where required. Conclusions were also drawn on the efficiency of the techniques to be applied for embankments with wood structures