1Department of Biology, Case Western Reserve University
Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.
Published December 19, 2014. Keywords: Plant Biology, Flax (Linum usitatissimum), Floral-dip, Plant transformation, Transgenic, Agrobacterium tumefaciens, Plant binary vector, Direct PCR testing
1Department of Plant Sciences, University of Oxford, 2School of Education of Vitoria-Gasteiz, University of the Basque Country (UPV/EHU), 3Biosciences, University of Exeter
This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.
Published December 19, 2014. Keywords: Plant Biology, Apoplast, apoplast washing fluid, plant leaves, infiltration-centrifugation, plant metabolism, metabolomics, gas chromatography-mass spectrometry
1Institut für Biochemie und Molekulare Biologie, Universität Ulm, 2Institut für Immunologie, Universitätsklinikum Ulm
Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.
Published December 16, 2014. Keywords: Stem Cell Biology, Intestinal stem cells, Drosophila melanogaster, aging, midgut dissection, transcriptome, fluorescence activated cell sorting
1Division of Neuropathology, Department of Pathology, Pathobiology Graduate Program, Johns Hopkins School of Medicine
This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.
Published December 16, 2014. Keywords: Physiology, TUNEL, fluorescence, skeletal muscle, DNA damage, image analysis, histology, SMA, motor neuron disease
1Cancer Biology, UCL Cancer Institute
Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.
Published December 16, 2014. Keywords: Cellular Biology, Image analysis, High-content analysis, Screening, Microscopy, Individual cell analysis, Multiplexed assays
1Department of Biology, Baylor University
The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.
Published December 13, 2014. Keywords: Cellular Biology, Saccharomyces cerevisiae, mRNA decay, mRNA stability, nonsense-mediated mRNA decay, mRNA half-life, transcription inhibition
1Cardiovascular Research Institute and Division of Cardiology, University of California San Francisco, 2Department for Biochemistry and Biophysics, University of California San Francisco, 3Max-Planck Institute for Heart and Lung Research
Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.
Published December 12, 2014. Keywords: Medicine, Zebrafish, heart, cardiovascular, dissection, adult, high-throughput
1Egg Safety and Quality Research Unit, USDA-Agricultural Research Service, 2Poultry Microbiological Safety and Processing Research Unit, USDA-Agricultural Research Service, 3Department of Biochemistry and Biophysics, Oregon State University, 4College of Public Health, University of Georgia, 5Department of Biological Sciences, Center for Microbial Genetics and Genomics, Northern Arizona University
A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.
Published December 10, 2014. Keywords: Molecular Biology, DNA extraction, poultry, environmental, feces, litter, semi-automated, microbiomics, qPCR
1Diagenode S.A., 2Diagenode Inc.
Methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research but they are laborious, costly, and time consuming. Here a commercially available robotic liquid handling system that automates chromatin immunoprecipitation for mapping in vivo protein-DNA interactions with limited amounts of cells is presented.
Published December 10, 2014. Keywords: Molecular Biology, Automation, chromatin immunoprecipitation, low DNA amounts, histone antibodies, sequencing, library preparation
1Department of Physical Medicine and Rehabilitation, Harvard Medical School, 2Cardiovascular Research Laboratory, Spaulding Hospital Cambridge
Cerebral perfusion is maintained across a range of pressures via cerebral autoregulation. However, characterizing autoregulation requires prominent pressure fluctuations at regulated frequencies. The described protocol will show how oscillatory lower body negative pressure can generate pressure fluctuations to provide data for projection pursuit regression for quantification of the autoregulatory curve.
Published December 10, 2014. Keywords: Medicine, cerebral blood flow, lower body negative pressure, autoregulation, sympathetic nervous system