Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.
1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California
Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.
Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.
An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.
Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization
1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine
Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).
1Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, 2Department of Internal Medicine, Washington University School of Medicine, 3Department of Pediatrics, Washington University School of Medicine
Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.
The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.
1Department of Cancer Genetics and Developmental Biology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3These authors contributed equally., 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC, 5Photography/Video Production, Multi-Media Services, BC Cancer Agency, 6Department of Medical Genetics, Life Sciences Institute,, University of British Columbia - UBC
This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).
1Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Donnelly Sequencing Centre, University of Toronto, 4Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, 5Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, 6Department of Pharmaceutical Sciences, University of Toronto
We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.
Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.
Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.
We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.
1Department of Applied Mathematics & Statistics, Stony Brook University, 2Computational Biology and Bioinformatics, Cold Spring Harbor Laboratory, 3Department of Molecular and Cell Biology, University of Texas at Dallas
Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.
We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.
Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.
Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.
1Centre of Excellence in Neuromics, CHUM Research Center and the Department of Medicine, Universite de Montreal, 2Center of Excellence in Neuromics, CHU Sainte Justine and CHUM Notre-Dame Research Centers, Universite de Montreal, 3Department of Medicine, Universite de Montreal
Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
1Department of Obstretrics & Gynaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Children's Health Research Institute
Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.
The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.
1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University
RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.
Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.
Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling
RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.
The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing β-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.
ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.
Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling
We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.
Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation
1Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, 2A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, 3Department of Biochemistry, National University of Singapore, Singapore
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
1Department of Molecular Genetics, University of Toronto, 2Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 3Department of Biochemistry, Research and Innovation Centre, University of Regina
Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.
We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.
We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.
Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography
We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.
1Dept. of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, 2Dept. of Biological Chemistry, University of California, Los Angeles, 3Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles
Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
1Department of Neurology, McKnight Brain Institute, University of Florida, 2Department of Entomology and Nematology, University of Florida, 3Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida, 4McKnight Brain Institute, Department of Neuroscience, Genetics Institute, Center for Translational Research on Neurodegenerative Diseases, and Center for Movement Disorders and Neurorestoration, University of Florida
We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.
A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.
A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica
We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).
Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides
This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.
Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping
The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.
The procedure for implementing a refractive index sensor for terahertz frequencies based on a grooved parallel-plate waveguide geometry is described here. The method yields a measurement of the refractive index of a small volume of liquid through monitoring of the shift in the resonant frequency of the waveguide structure
This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.
Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.
1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh
The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine
Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.
Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy
A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.
Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)
The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.
Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.
The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.