Methods in molecular biology
-
This chapter discusses two microfluidic-based approaches for early sepsis detection that achieve a higher accuracy than traditional blood culture analysis. Patient blood samples were included in this work to validate the performance of our chips in diagnosing sepsis. The single-parameter chip demonstrated the increased accuracy if using CD64 as a biomarker for sepsis detection compared with C-reactive protein (CRP) and procalcitonin (PCT) when applied alone. ⋯ The combined panel was also able to detect culture-negative patients and provided a faster diagnosis. Besides, microfluidics has advantages of less time consuming, easier to manufacture, less sample loading, less complex, and portable. Therefore, our approach is of great potential to become a bedside sepsis detection method.
-
In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 h.
-
CRISPR-associated nuclease (Cas) has been widely applied to modify the genomes of various cell types. As RNA-guided endonucleases, Cas enzymes can target different genomic sequences simply by changing the guide sequence of the CRISPR RNA (crRNA) or single guide RNA (sgRNA). Recent studies have demonstrated that DNA-RNA chimeric crRNA or sgRNA can efficiently guide the Cas9 protein for genome editing with reduced off-target effects. This chapter aims to describe a procedure for using chimeric RNA to modify the genomes of mammalian cells.
-
Whole-genome bisulfite sequencing (WGBS) is a popular method for characterizing cytosine methylation because it is fully quantitative and has base-pair resolution. While WGBS is prohibitively expensive for experiments involving many samples, low-coverage WGBS can accurately determine global methylation and erasure at similar cost to high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assays (ELISA). ⋯ In addition to describing a WGBS library construction and quantitation approach, here we detail computational methods to predict the accuracy of low-coverage WGBS using empirical bootstrap samplers and theoretical estimators similar to those used in election polling. Using examples, we further demonstrate how non-independent sampling of cytosines can alter the precision of error calculation and provide methods to improve this.
-
Allergic asthma is characterized by airway hyperresponsiveness, remodeling, and reversible airway obstruction. This is associated with an eosinophilic inflammation of the airways, caused by inhaled allergens such as house dust mite or grass pollen. The inhaled allergens trigger a type-2 inflammatory response with the involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high immunoglobulin E (IgE) antibody production by B cells and mucus production by airway epithelial cells. ⋯ Finally, mice are challenged by intranasal allergen administrations. We will also describe the protocols as well as the most important readout parameters for the measurements of invasive lung function, serum immunoglobulin levels, isolation of bronchoalveolar lavage fluid (BALF), and preparation of cytospin slides. Moreover, we describe how to perform ex vivo restimulation of lung single-cell suspensions with allergens, flow cytometry for identification of relevant immune cell populations, and ELISAs and Luminex assays for assessment of the cytokine concentrations in BALF and lung tissue.