Project description:Untargeted multi-omics analysis of plasma is an emerging tool for the identification of novel biomarkers for evaluating disease prognosis and for a better understanding of molecular mechanisms underlying human disease. The successful application of metabolomic and pro-teomic approaches relies on reproducibly quantifying a wide range of metabolites and proteins. Herein, we report the results of untargeted metabolomic and proteomic analyses from blood plasma samples following analyte extraction by two frequently used solvent systems: chloro-form/methanol and methanol-only. Whole blood samples were collected from participants (n=6) at University Hospital Sharjah (UHS) hospital, then plasma was separated and extracted by two methods i. methanol precipitation and, ii. 4:3 methanol:chloroform extraction. The coverage and reproducibility of the two methods were assessed by ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). The study revealed that metabolite extraction by methanol-only showed greater reproducibility for both metabolomic and proteomic quantifications than did methanol/chloroform, while yielding similar peptide coverage. However, coverage of extracted metabolites was higher with the methanol/chloroform precipitation.
Project description:As the importance of transcriptional variation and regulation for Plasmodium becomes more apparent, advances for non-falciparum species are hindered by our reliance on natural infections to study parasite biology. Untargeted transcriptomic research is also complicated by low parasite densities and high proportions of human genetic material, highlighting the need for optimized sample processing protocols. In this study, we used a P. knowlesi culture diluted in whole blood as a mock P. vivax natural infection to compare white blood cell, rRNA-, and globin depletion methods and RNA-seq library preparation kits to create an optimized protocol for low-volume sample processing.
Project description:Gene expression profiling by high-throughput sequencing determines changes in gene expression only at steady state but prevents our understanding of the underlying gene expression kinetics. Here, we describe a protocol that combines metabolic RNA labeling with thiol-specific chemical nucleoside conversion to determine the stability of polyadenylated RNA transcripts. And we provide a targeted mRNA 3 end library preparation protocol that enable to robustly determine the stability even of RNA transcripts that escape robust detection in untargeted libraries. The described methods enable cost-effective insights into the kinetics underlying steady-state gene expression in order to study the mechanisms underlying the regulation of gene expression at a transcript-specific and genomic scale.
Project description:<p><strong>INTRODUCTION:</strong> The extraction solvent mixtures were optimized for untargeted metabolomics analysis of microbial communities from two laboratory scale activated sludge reactors performing enhanced biological phosphorus removal (EBPR).</p><p><strong>OBJECTIVE:</strong> To develop a robust and simple analytical protocol to analyse microbial metabolomics from EBPR bioreactors.</p><p><strong>METHODS:</strong> Extra- and intra-cellular metabolites were extracted using five methods and analysed by ultraperformance liquid chromatography mass spectrometry (UPLC-MS).</p><p><strong>RESULTS:</strong> The optimal extraction method was biomass specific and methanol:water (1:1 v/v) and methanol:chloroform:water (2:2:1 v/v) were chosen, respectively, for each of the two different bioreactors.</p><p><strong>CONCLUSION:</strong> Our approach provides direct surveys of the metabolic state of PAO-enriched EBPR communities, showing that extraction methods should be carefully tailored to the microbial community under study</p>