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Reproducible Quantitative PCR of Mitochondrial and Nuclear DNA Copy Number Using the LightCycler
Time of Update: 2021-02-22
Recent developments inPCR fluorimetry have allowed for quick quantification of target molecules. Before the invention of fluorimetric quantitative PCR, researchers who wanted to quantify the amount
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Preparation of Chromatin Assembly Extracts from Xenopus Oocytes
Time of Update: 2021-02-22
The majority ofDNA in eukaryotic cells is packaged by histones and many poorly characterized nonhistone proteins to form a dynamic structure known as chromatin.
Moreover, histones, via protein-protein interactions or by playing an architectural role, can facilitate or inhibit the transcriptional activation process ( 1 , 2 ).
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Analysis of Chromatin by Scanning Force Microscopy
Time of Update: 2021-02-22
The most widely used probe microscopy for biological applications, the scanning force microscope (SFM), also known as the atomic force microscope, is capable of imaging samples under very mild conditions (for recent reviews,see refs.
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Extraction of Yeast Lipids
Time of Update: 2021-02-22
Quantitative extraction of lipids from the tissue or microorganism of choice is key to their subsequent analysis. In this chapter, we describe a simple and rapid protocol that relies on glass bead di
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Preparation of Culture Filtrate Proteins from Mycobacterium tuberculosis
Time of Update: 2021-02-22
Culture filtrates obtained by in vitro cultivation ofMycobacterium tuberculosis have been studied for more than 20 years to identify and characterize proteins of immunological relevance.
Culture filtrate preparations have been shown to induce a protective immune response in mice and guinea pigs by several groups ( 1 – 4 ).
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Food-Grade Corynebacteria for Enzyme Production
Time of Update: 2021-02-21
Using genetic manipulation techniques, several corynebacteria strains expressing genes coding for hydrolytic enzymes or proteins have been constructed in different laboratories.
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The Use of Monoclonal Antibodies and Lectins to Identify Changes in Viral Glycoproteins That are Influenced by Glycosylation: The Case of Human Respiratory Syncytial Virus Attachment (G) Glycoprotein
Time of Update: 2021-02-21
Given the role of the G glycoprotein in RSV antigenicity, we also describe procedures based on Western blotting to determine the effect of G protein glycosylation changes on reactivity with human sera.
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Comparative Genome Analysis of Clostridium difficile Using DNA Microarrays
Time of Update: 2021-02-21
However, to fully exploit this opportunity, robust phylogenetic methods on a diverse set of characterised strains are required to provide a reference evolutionary framework to studyC.
Traditional phylogenetic classification of bacteria to study evolutionary relatedness is based on the characterisation of a limited number of genes, rRNA or signature sequences.
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Application of Microwave-Assisted Extraction for the Analysis of Dithiocarbamates in Food Matrices
Time of Update: 2021-02-21
Quantification is based on external standard calibration curves made with either standard solutions of individual DMDTCs (thiram, ziram) or EBDTCs (maneb, zineb, mancozeb), which are processed as the field samples, or using standard solutions of CS 2 made in isooctane.
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Neurosporaxanthin Production by Neurospora and Fusarium
Time of Update: 2021-02-21
The orange pigmentation of the ascomycete fungiNeurospora andFusarium is mainly due to the accumulation of neurosporaxanthin, a carboxylic apocarotenoid whose possible biotechnological applications have not been investigated.
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Fit-for-Purpose Quenching and Extraction Protocols for Metabolic Profiling of Yeast Using Chromatography-Mass Spectrometry Platforms
Time of Update: 2021-02-21
Methods to sample the exometabolome and to quench metabolism and extract intracellular metabolites for the model eukaryoteSaccharomyces cerevisiae are presented here.
For endometabolome studies, fast quenching of intracellular metabolism is performed using a 60:40 (v/v) methanol:aqueous ammonium hydrogen carbonate solution at –48�C.
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An Approach Combining Rapid cDNA Amplification and Chemical Synthesis for the Identification of Novel, Cathelicidin-Derived, Antimicrobial Peptides
Time of Update: 2021-02-21
Host defense in mammals involves a cooperative effort by a variety of antimicrobial peptides ( 1 ), which are produced by both phagocyte precursors and some epithelia. They are called on to combat in
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Measurement of Cytokines in Synovial Fluid
Time of Update: 2021-02-21
Over the last 20 years, advances in the knowledge of cytokine biology have brought a new dimension to the understanding of pathogenic events in human chronic arthritides, showing promise for future therapeutic approaches.
It is beyond the scope of this chapter to describe in detail the available evidence concerning the role of cytokines in these diseases.
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Quantification of Host and Phage mRNA Expression During Infection Using Real-Time PCR
Time of Update: 2021-02-21
Real-time, or quantitativePCR , is a valuable technique useful in bacteriophage research to quantify the abundance of phage or host gene transcripts.
This chapter should also be useful to those wishing to amplify genes from other types of templates such as metagenomic DNA or RNA extracted either from filtered samples or from agarose gels.
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Generation of Astaxanthin Mutants in Xanthophyllomyces dendrorhous Using a Double Recombination Method Based on Hygromycin Resistance
Time of Update: 2021-02-21
In this chapter is described a method that allows the double gene deletions of the two copies of a gene from a diploid organism, a wild-type strain of theXanthophyllomyces dendrorhous yeast, using hygromycin B resistance as the only selection marker.
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Animal Models in Drug Development for MRSA
Time of Update: 2021-02-21
In this way animal models of infection can be a highly valuable and predictive bridge between in vitro drug discovery and early clinical evaluation.
This chapter will describe the advantages, methods, and outcome measurements of the rodent models most used in drug discovery forS.
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In Vivo Assays of Langerhans Cell Migration
Time of Update: 2021-02-21
The skin is an immunologically active tissue; the integrated skin immune system comprises epidermal Langerhans cells (LC), dermal dendritic cells (DC), tissue macrophages, mast cells, and T lymphocyt
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Measurement of Tumor Necrosis Factor (TNF) Lysis of Adenovirus-Infected Cells
Time of Update: 2021-02-21
To minimize or prevent the spread of an acute virus infection, the antiviral immune response must detect and lyse virus-infected cells before virus replicates or is released from the host cell.
The immune response has developed both innate and specific responses to meet this objective.
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Bacteriocin Production Process by a Mixed Culture System
Time of Update: 2021-02-21
Antimicrobial proteins and peptides produced by bacteria, termed bacteriocin, are widely acknowledged to be important contributors to those organisms that survive dominate or die in microbial ecosystem such as our food supply or digestive tract.
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Absolute and Relative Quantification of mRNA Expression (Transcript Analysis)
Time of Update: 2021-02-21
In the next section, we concentrate on providing a detailed protocol for the validation of these data using quantitative reverse transcriptase-polymerase chain reaction (qRT- PCR ).
The protocol provides suggested examples of hardware, software, and consumables/reagents required to perform these experiments.
