Methods in molecular biology最新文献

Immediately after birth, mammals are largely colonized by microorganisms, with the gastrointestinal tract being the most commonly colonized organ. Over the years, several studies have shown that the intestinal microbiota is important for various physiological functions of the host. Gnotobiotic animal models are frequently used to better understand how the microbiota influences health and disease scenarios. Among gnotobiotic models, germ-free (GF) animals were first used in 1895, but it was not until 60 years later that germ-free colonies were suitable for large-scale experiments. The use of GF mice is an interesting and rich tool for studying the microbiome. However, their maintenance is a complex process that needs to be done carefully. In this chapter, we describe step by step how to manage and manipulate the gut microbiota of GF mice.

Remarkable progress in basic, translational, and clinical cancer research has been observed during the last decade. This has opened possibilities for the development of novel diagnostics and therapeutic approaches and created opportunities for personalized medicine. Cancer biomarkers are key players in human cancer progression, both peripheral and at the site of tumor. Through reliable techniques detecting biomarkers, cancer can thus be predicted, diagnosed and progression and response to therapy can be followed. Multiplex analysis of biomarkers in small blood volumes allows for rapid quantification of large number of circulating analytes. The Luminex technique allows multiple biomarkers to be measured simultaneously in small volumes and provides a convenient and sensitive tool for the detection of large number of extracellular secreted biomarkers to be used in prediction and therapy prognosis in cancer. The technique is based on so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed microbeads have monoclonal antibodies directed against the biomarker of interest and allow simultaneous detection of up to hundreds of biomarkers in a dual-laser flow analyzer. Biomarkers can be detected in serum- and plasma samples as well as in cell culture supernatants from in vitro cultured and stimulated cells, e.g., peripheral blood mononuclear cells (PBMC) or cancer cell lines.The need for robust detection of biomarkers for prediction as well as outcome of cancer therapy progression is of great importance. This chapter describes the Luminex technique for detection of biomarkers associated with cancer by magnetic bead sandwich immunoassay, with focus on some important pre-analytic factors, e.g., cell separation and cryopreservation and thawing of PBMC that may affect the outcome of detection of biomarkers. The Luminex technique is thus one way to discover biomarkers to predict, prognose, and improve clinical outcome of cancer.

The Luminex multiplex bead immunoassay enables the detection of more than 100 analytes in a sample at a time. This method utilizes magnetic beads coated with antibodies of interest to measure multi-analytes simultaneously. Here, we describe a detailed protocol for multi-analyte detection of two proteins, fatty acid binding protein 1 (FABP1) and fibroblast growth factor 19 (FGF19), both related to fat metabolism, in cell lysates of HepG2 hepatocytes.

Animal models, particularly murine protocols on physical exercise, are widely used to investigate physiological adaptations and to study, prevent, and treat chronic non-communicable diseases. Controlled treadmill running enables precise manipulation of intensity and duration, overcoming limitations of voluntary wheel running for modeling inflammation. An incremental-speed treadmill test to fatigue disrupts homeostasis and acts as an acute inflammatory stimulus, eliciting local skeletal muscle and systemic immune responses. This chapter presents a standardized physical exercise protocol in mice and subsequent tissue collection, combined with intravital microscopy of skeletal muscle microcirculation and in vitro neutrophil migration assays, to quantify leukocyte recruitment and activation as a versatile model of exercise-induced inflammation.

Although high-throughput sequencing methods have greatly improved over the last few years, direct sequencing remains unfeasible when DNA quantity or quality is limited. In such instances, various whole genome or metagenome amplification (WGA) techniques can generate sufficient DNA for multiple analyses, albeit with some amplification bias. Competent WGA analysis is typically evaluated by sequence coverage, assessed through two key parameters: depth, referring to the number of reads containing each nucleotide, and breadth, indicating the proportion of nucleotides in the consensus sequence relative to the original sequence length at the obtained depth. Adequate coverage is essential for detailed genomic analysis and the detection of population variants, copy number variations (CNVs), and structural variants (SVs). This chapter outlines a pipeline for analyzing Illumina sequencing data of amplified samples compared to non-amplified samples to assess the performance of various WGA methods, starting from raw sequences.

Respiratory syncytial virus (RSV) infection continues to be a significant burden on public health care systems and is a global health concern. Whole genome sequencing (WGS) provides a useful tool to better understand the viral transmission and emerging mutations that may impact antibody treatments, antiviral drug sensitivity, and vaccine effectiveness. Here, we describe a rapid and sensitive protocol for sequencing clinical samples of both human RSV-A and RSV-B viruses based on the Oxford Nanopore Technology (ONT) sequencing platform. It involves long amplicon generation by setting up two one-step multiplex reverse-transcription polymerase chain reactions (mRT-PCR) for each sample, library preparation with the ONT rapid barcoding kit and NGS data analysis with the ARTIC pipeline.

The genomic analysis of circulating tumor cells (CTCs) can help us identify their specific characteristics and reveal intratumor heterogeneity while also reducing the need for invasive sampling. However, studying single CTCs can be challenging, as whole-genome amplification (WGA) is needed to obtain enough genetic material for high-throughput sequencing. Here, we present a protocol for isolating single CTCs from mouse xenografts, followed by WGA using the Ampli1WGA Plus kit.

Fourier transform infrared spectroscopy (FTIR) is a type of spectroscopy known as "vibrational." Widely used for protein analysis, it also provides a wealth of information on the structure and stability of nucleic acids. Its advantages are that it requires a small amount of sample and that it is easy to vary experimental conditions (pH, temperature, ionic strength, and composition). While numerous reviews describe how to analyze DNA structure alone or in the presence of proteins using FTIR, analyses of RNA are scarce. Nevertheless, RNAs are important factors involved in a multitude of roles in the cell. In this chapter, we present applications of FTIR to analyze RNA structure and how proteins or modified nucleosides may change this structure.

Laser microdissection (LMD) and whole genome amplification (WGA) are powerful techniques that integrate molecular and histological approaches to enable the precise selection of a minimal number of virus-infected cells-down to near single-cell resolution-and the subsequent generation of whole viral genomes with minimal host DNA interference. This chapter presents a detailed protocol for LMD and near single-cell WGA, specifically optimized for the recovery and sequencing of viral genomes from formalin-fixed paraffin-embedded (FFPE) tissues. The method allows for the targeted isolation of infected cells, thereby reducing host genomic background and enhancing the detection of pathogen-specific signals for downstream next-generation sequencing (NGS). The protocol includes steps for tissue section preparation, cell isolation via LMD, DNA extraction using the PicoPure DNA Extraction Kit, and unbiased genome amplification using the SeqPlex DNA Amplification Kit-ensuring high-quality nucleic acid recovery suitable for NGS workflows.

Alternative splicing is a crucial post-transcriptional regulatory mechanism that generates multiple protein isoforms from a single gene, substantially increasing the coding capacity and proteome diversity of a genome. This process is particularly critical in regulating the activity of interleukin genes, where alternative splicing contributes to the functional diversity of these important immune system molecules and affects their production, function, and receptor interactions. While numerous studies have established the connection between aberrant alternative splicing and various diseases, including cancers and autoimmune disorders, the function and regulation of many splice variants remain poorly understood. Here, we describe a cost-effective and reliable method for analyzing alternative splicing patterns in interleukin genes using semi-quantitative RT-PCR and densitometry analysis. This method enables the simultaneous identification and quantification of multiple splice variants in a single PCR reaction, offering advantages over real-time RT-PCR approaches that require specific primer sets for each variant. This protocol involves RNA extraction from tissue culture cell lines or tissue samples, reverse transcription, RT-PCR, and subsequent analysis using freely available software for densitometry. We demonstrate the utility of this approach through two distinct examples with different alternative splicing patterns. While less sensitive than real-time RT-PCR or radioactive methods, this technique provides a robust, accessible, and widely accepted approach for investigating alternative splicing patterns in interleukin genes, contributing to our understanding of cytokine biology and its role in health and disease.