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Protein Blotting: Immunoblotting
Duojiao Ni, Peng Xu, Diviya Sabanayagam, Sean R. Gallagher
Immunoblotting (also referred to as western blotting) uses antibodies to probe for a specific protein in a sample bound to a membrane. Typically, a protein sample is first size separated via electrophoresis (e.g., SDS PAGE). However, antibodies used for specific protein detection are restricted by
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Conventional Light Microscopy
Eric S. Cole
In this molecular day and age, microscopy seems to be a neglected field of instruction. Too often professors, who themselves are strangers to the use of the light microscope, may hurry through a laboratory exercise designed to familiarize students with its uses. This chapter is designed to serve as
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Submitting a Sequence to GenBank
Wei‐Jen Chang, Kassandra E. Zaila, Thomas W. Coppola
In the post‐genomic era, more and more research projects involve the generation of molecular sequence data. How should these newly obtained DNA/protein sequences be analyzed, and how should they be prepared for submission to sequence databases? In this unit, we provide guidelines and a flowchart to
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Whole‐Genome Sequencing Analysis Using Next‐Generation Sequencing Data
Chi Kent Ho, Xiaohui Cui, Sharon Grubner, Christopher A. Larson, Ying Wei, Paul K. Flook
Next‐generation sequencing (NGS) technologies have revolutionized the biosciences and become invaluable to the discovery of gene function and its involvement in disease conditions. The fast pace of innovation in NGS technologies has enabled the production of huge volumes of sequence data at
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CLIP‐seq to Identify KSHV ORF57‐Binding RNA in Host B Cells
Yanping Ma, Poching Liu, Vladimir Majerciak, Jun Zhu, Zhi‐Ming Zheng
Kaposi's sarcoma‐associated herpesvirus (KSHV), a human gamma ‐herpesvirus, is etiologically linked to the development of several malignancies, mainly Kaposi's sarcoma. Expressed as an early viral protein, KSHV ORF57 is essential for lytic replication and virion production. ORF57 selectively binds
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Biochemical Analysis of Microbial Rhodopsins
Julia A. Maresca, Jessica L. Keffer, Kelsey J. Miller
Ion‐pumping rhodopsins transfer ions across the microbial cell membrane in a light‐dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodopsin identification in environmental and genomic sequence data sets, in vitro
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Detection of Papillomavirus Gene Expression Patterns in Tissue Sections
Heather Griffin, John Doorbar
Molecular events during the papillomavirus life cycle can be mapped in infected tissue biopsies using antibodies to viral and cellular gene products, or by in situ hybridization approaches that detect viral DNA or viral transcription products. For proteins, ease of immunodetection depends on
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Using Organotypic Epithelial Tissue Culture to Study the Human Papillomavirus Life Cycle
Denis Lee, Kathryn Norby, Mitchell Hayes, Ya‐Fang Chiu, Bill Sugden, Paul F. Lambert
Human papillomaviruses (HPVs) are small double‐stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a
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PA‐seq for Global Identification of RNA Polyadenylation Sites of Kaposi's Sarcoma–Associated Herpesvirus Transcripts
Ting Ni, Vladimir Majerciak, Zhi‐Ming Zheng, Jun Zhu
Kaposi's sarcoma–associated herpesvirus (KSHV) is a human oncovirus linked to the development of several malignancies in immunocompromised patients. Like other herpesviruses, KSHV has a large DNA genome encoding more than 100 distinct gene products. Despite being transcribed and processed by
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Mimiviruses: Replication, Purification, and Quantification
Jônatas Santos Abrahão, Graziele Pereira Oliveira, Lorena Christine Ferreira da Silva, Ludmila Karen dos Santos Silva, Erna Geessien Kroon, Bernard La Scola
The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower water sample collected during an outbreak of pneumonia in a hospital in Bradford,
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Genomic DNA Isolation from Maize (Zea mays) Leaves Using a Simple, High‐Throughput Protocol
Kristen A. Leach, Paula C. McSteen, David M. Braun
A simple, robust, inexpensive, high‐throughput method for isolating genomic DNA from maize ( Zea mays ) leaf tissues is described. The DNA obtained using this extraction protocol is suitable for polymerase chain reaction (PCR) genotyping, which can be employed for the identification of alleles in
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Protein Interaction Profile Sequencing (PIP‐seq) in Plants
Stephen J. Anderson, Matthew R. Willmann, Brian D. Gregory
RNA secondary structure and RNA‐protein interactions are necessary for maintaining biological functionality and regulatory mechanisms within eukaryotic transcriptomes. Determining the structural characteristics and protein‐bound sites of RNA molecules has therefore become a major research objective
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Preparation of Rice Plant Genomic DNA for Various Applications
Lian Zhou, Rongxin Shen, Xingliang Ma, Heying Li, Gousi Li, Yao‐Guang Liu
Research on plant molecular biology, genetic engineering, and crop molecular breeding involves various manipulations of plant genomic DNAs (gDNAs). These studies require preparing gDNAs from plant materials using suitable methods according to the yield, intactness, and purity of the resultant gDNA
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Small RNA Extraction and Detection in Rice (Oryza sativa)
Xianwei Song, Xiaofeng Cao
Rice ( Orzya sativa ) small RNAs regulate almost all biological processes and agronomic traits. Characterization and dissection of small RNA functions have become an important part of rice functional genomic research. Isolation of small RNAs of desirable quality is essential for a variety of
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Chromosome Preparation in Rice (Oryza sativa)
Kai Wang, Weichang Yu
Chromosomes are the carriers of genetic material in biological organisms. Each chromosome has three essential components: a centromere, telomeres, and chromosome arms. Chromosome preparation is the basic technique for studies in cytogenetics, including the analysis of chromosomal behavior,
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In Situ Hybridization in Rice (Oryza sativa)
Kai Wang, Weichang Yu
Fluorescence in situ hybridization (FISH) is widely used in cytogenetics to determine the localization of DNA sequences on target chromosomes, to provide visible information regarding the physical position of DNA sequences, to determine the abundance and distribution of repetitive sequences that
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Artificial Chromosomes in Rice (Oryza sativa)
Chunhui Xu, Weichang Yu
Chromosomes are the carriers of genetic material in biological organisms. Each chromosome has three essential components: a centromere, telomeres, and origins of replication. The understanding of the essential structural and functional organization of chromosomes has made it possible to produce
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Chromosome Preparation for Cytogenetic Analyses in Arabidopsis
Terezie Mandáková, Martin A. Lysak
Despite its small nuclear genome and minute chromosomes, the model plant Arabidopsis thaliana has become a well established model in the field of plant cytogenetics. Since 2000, most cytogenetic and epigenetic approaches have been developed for this species and its congeners. In this chapter, we
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Artificial Chromosome Preparation in Arabidopsis
Minoru Murata
In Arabidopsis thaliana , various attempts have been made to create artificial chromosomes as a new tool for cytological and genetic analyses. However, most of the efforts have been unsuccessful until recently. Most eukaryotic chromosomes are linear, and therefore the Arabidopsis artificial
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Purification of Translating Ribosomes and Associated mRNAs from Soybean (Glycine max)
Norma A. Castro‐Guerrero, Yaya Cui, David G. Mendoza‐Cozatl
Cell identity and function are largely determined by specific gene expression patterns and ultimately by the proteome. Current high‐throughput sequencing technologies offer the possibility of quantifying gene expression at high resolution, with minimum input and without the constraints of
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Maize (Zea mays) Hi‐II Transformation via Agrobacterium‐Mediated T‐DNA Transfer
Hyeyoung Lee, Zhanyuan J. Zhang
Genetic transformation of maize via Agrobacterium tumefaciens is still more art than science, with different researchers achieving substantially different transformation results. This article describes our advanced Agrobacterium ‐mediated transformation system in Hi‐II maize. The system utilizes
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Generation of Soybean (Glycine max) Transient Transgenic Roots
Katalin Tóth, Josef Batek, Gary Stacey
Legumes—because of their nitrogen‐fixing capacity—have both ecological and agronomic importance, and are also the major plant protein source for animal consumption. The model legume species are Lotus japonicus , Medicago truncatula , and soybean ( Glycine max ). These species have sequenced genomes
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Soluble Sugar and Starch Extraction and Quantification from Maize (Zea mays) Leaves
Kristen A. Leach, David M. Braun
An easy‐to‐perform protocol for isolating and quantifying soluble sugars (sucrose, glucose, and fructose) and starch from maize (Zea mays ) leaf tissue is described. The method has been optimized to extract non‐structural carbohydrates (NSC) from frozen, finely ground tissue in a
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RNA‐Seq Library Construction Methods for Transcriptome Analysis
Nathan J. Bivens, Mingyi Zhou
Next‐generation sequencing (NGS) technologies have revolutionized the study of genomics with an ever‐expanding list of applications. RNA‐Seq has emerged as a powerful method, applying transcriptome analysis to a wider range of organisms—most significantly, non‐model organisms lacking prior genomic
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Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana
Erica D. LaMontagne, Carina A. Collins, Scott C. Peck, Antje Heese
Cellular membranes define the boundaries between organelles and the cytosol or the extracellular environment, thus providing functional separation between subcellular compartments. In addition, membranes assist in a diverse range of cellular functions, including serving as signaling platforms,
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A Method for Sectioning and Immunohistochemical Analysis of Stem Cell–Derived 3‐D Organoids
Luke A. Wiley, David C. Beebe, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker
This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell–derived 3‐D organoids. Specifically, we describe a method to embed iPSC‐derived retinal cups in low‐melt agarose, acquire thick sections using a vibratome tissue slicer, and perform
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Production of 3‐D Airway Organoids From Primary Human Airway Basal Cells and Their Use in High‐Throughput Screening
Marc Hild, Aron B. Jaffe
The ability of human airway basal cells to serve as progenitor cells in the conducting airway makes them an attractive target in a number of respiratory diseases associated with epithelial remodeling. This unit describes a protocol for the culture of ‘bronchospheres’, three‐dimensional (3‐D)
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Isolation of Human Amnion Epithelial Cells According to Current Good Manufacturing Procedures
Roberto Gramignoli, Raghuraman C. Srinivasan, Kristina Kannisto, Stephen C. Strom
Different cell types can be isolated from human placental tissues, and some have been reported to retain phenotypic plasticity and characteristics that make them a promising source of cells for regenerative medicine. Among these are human amnion epithelial cells (hAECs). Adoption of current good
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Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay
Filomena S.G. Silva, Irina G. Starostina, Vilena V. Ivanova, Albert A. Rizvanov, Paulo J. Oliveira, Susana P. Pereira
The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for
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Investigating the Effects of Particulate Matter on House Dust Mite and Ovalbumin Allergic Airway Inflammation in Mice
Alejandro R. Castañeda, Kent E. Pinkerton
Particulate matter (PM), a component of air pollution, has been shown to enhance allergen‐mediated airway hypersensitivity and inflammation. Surprisingly, exposure to PM during the sensitization to allergen is sufficient to produce immunological changes that result in heightened inflammatory effects
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Multielectrode Array (MEA) Assay for Profiling Electrophysiological Drug Effects in Human Stem Cell‐Derived Cardiomyocytes
Mike Clements
More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Human stem cell‐derived cardiomyocytes (hSC‐CMs) provide a potential model for the development of superior assays for preclinical drug safety screening. One such
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