Methods in molecular biology
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Detection of Differential DNA Methylation Under Stress Conditions Using Bisulfite Sequence Analysis.
DNA methylation is the most important epigenetic change affecting gene expression in plants grown under normal as well as under stress conditions. Therefore, researchers study differential DNA methylation under distinct environmental conditions and their relationship with transcriptome abundance. Up to date, more than 25 methods and techniques are available to detect DNA methylation based on different principles. ⋯ This technique allows a single nucleotide resolution of 5-methylcytosine on a genome scale. WGBS technique workflow involves DNA fragmentation, processing through end blunting, terminal A(s) addition at 3' end and adaptor ligation, bisulfite treatment, PCR amplification, sequencing libraries and assembling, and finally alignment with the reference genome and data analysis. Despite the fact that WGBS is more reliable than the conventional clone-based bisulfite sequencing, it is costly, requires large amount of DNA and its output data is not easily handled.
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Rapid diagnostic methods for fungal infections are long awaited and are expected to improve outcomes through early initiation of targeted antifungal therapy. T2Candida panel is a novel qualitative diagnostic platform that was recently approved by the US Food and Drug Administration (FDA) for diagnosis of candidemia with a mean time to species identification of less than 5 h. ⋯ By combining magnetic resonance with molecular diagnostics, T2Candida panel amplifies DNA and detects the amplified product by amplicon-induced agglomeration of supermagnetic particles and T2 Magnetic Resonance (T2MR) measurement. Here we describe the materials and methods needed to diagnose candidemia with the T2Candida panel.
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Cell signaling and functions heavily rely on post-translational modifications (PTMs) of proteins. Their high-throughput characterization is thus of utmost interest for multiple biological and medical investigations. ⋯ However, the large and complex datasets produced pose multiple data interpretation challenges, ranging from spectral interpretation to statistical and multivariate analyses. Here, we present a typical workflow to interpret such data.
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Comparative profiling proteomics experiments are important tools in biological research. In such experiments, tens to hundreds of thousands of peptides are measured simultaneously, with the goal of inferring protein abundance levels. ⋯ Previously we have reported the non-normal distribution of SILAC datasets, and demonstrated the permutation test to be a superior method for the statistical evaluation of non-normal peptide ratios. This chapter outlines the steps and the R scripts that can be used for performing permutation analysis with false discovery rate control via the Benjamini-Yekutieli method.
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Extracellular vesicle (EV)-associated RNAs (EV-RNA) are under intense investigation due to their potential role in health and disease. Several approaches are currently employed to isolate blood-derived EVs for RNA analysis, most of which are either time-consuming and expensive, such as methods based on EVs physical properties (ultracentrifugation and Optiprep density gradient), or also copurify blood contaminants, mostly protein aggregates and immune complexes, (such as chemical precipitation). In addition, there is a lack of standardized protocols for the extraction of EV-RNA and very little consensus on the technological platforms and normalization tools for assessing the expression levels of different RNA species. ⋯ In this book chapter we propose a protocol that might overcome some of the abovementioned issues through antibody-based isolation of blood-derived EVs followed by extraction and expression analysis of small-RNA species (miRNA) by reverse transcriptase quantitative PCR (RT-qPCR). The advantages of immunoaffinity approaches over other isolation methods are multiple and include: (1) the selective enrichment of specific EV subpopulations with restricted tissue/cell origin, (2) reduction of matrix effects and blood contaminants that may confound miRNA profiling from complex biological fluids and (3) easy coupling to conventional quantitative assays (e.g., RT-qPCR). In conclusion, we describe a protocol for standard enrichment and quantitative analysis of EV-miRNAs from blood and we warrant for technological improvements, such as the use of novel biomaterials, surface chemistries, binding agents and assay/sensor design that may further improve it.