An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci
1Center for Oral Biology, University of Rochester Medical Center, 2State Key Laboratory of Oral Diseases, Sichuan University, 3Department of General Medicine, Glostrup Hospital, Glostrup, Denmark, 4Department of Microbiology and Immunology, University of Rochester Medical Center
Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.
We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.
A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.
Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP
A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.
An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.
Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
1Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, 2Fluid Dynamics Group, CSIRO Materials Science and Engineering, 3Swinburne University of Technology, Faculty of Engineering and Industrial Sciences
We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.
High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
1The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 2Center for Reproductive Sciences, University of California San Francisco, 3Department of Urology, University of California San Francisco, 4Department of Cell and Tissue Biology, University of California San Francisco, 5Fluidigm Corporation, Fluidigm Corporation, 6Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, 7UCSF - Helen Diller Family Comprehensive Cancer Center, University of California San Francisco
Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.
Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format
Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.
Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer
1Department of Medicine, Baylor College of Medicine, 2Division of Diabetes, Endocrinology & Metabolism, Diabetes & Endocrinology Research Center, Baylor College of Medicine, 3Department of Molecular & Cellular Biology, Baylor College of Medicine
We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.
Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.
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.
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.
Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.
γ-Herpesviruses (γ-HVs) establish life-long persistency in their host. Infection of mice with γ-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of γHVs. This protocol describes the detection and quantitation of γHV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.
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.
In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.
1Department of Pediatrics, University of Texas Southwestern Medical Center, 2Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Pediatrics and Microbiology, University of Texas Southwestern Medical Center
A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.
Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.
The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.
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.
Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods
1Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran, 2Department of Endocrinology & Female Infertility, Reproductive Biomedicine Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.
The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.
1Department of Laboratory Medicine & Pathobiology, University of Toronto, 2Division of Urology, Sunnybrook Health Sciences Centre, Toronto, Canada, 3Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, Canada, 4Biological Sciences, Sunnybrook Research Institute
Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.
Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.
This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.
We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.
A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium
1Department of Medicine, Division of Cardiology, Emory University, 2Coulter Department of Biomedical Engineering, Georgia Tech and Emory University, 3Department of Bioinspired Science, Ewha Womans University
This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.
Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.
A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.
1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 2Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 3Shared Resource-DNA/RNA Peptide, Beckman Research Institute of City of Hope
Several 2’-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.
In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.
In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.
Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes
We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.
The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.
We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.
Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.
The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.
A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.
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.
We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.
1Wisconsin Water Science Center, United States Geological Survey, 2University of Wisconsin – Madison, 3Agricultural Research Service, United States Department of Agriculture, 4Alaska Science Center, United States Geological Survey
Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.
Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model
Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.
1Laboratorio de Investigación y Desarrollo (LID), Universidad Peruana Cayetano Heredia, 2Bloomberg School of Public Health, Johns Hopkins University, 3Laboratorio de Diagnostico Molecular, Facultad de Medicina, University of Concepcion,Chile, 4University of California San Diego School of Medicine
A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children’s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.
An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.
1Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin, 2Institute for Chemistry and Biochemistry, Free University Berlin, 3Laboratory for Functional Genomics Charité (LFGC), Charité - Universitätsmedizin Berlin, 4Comprehensive Cancer Center Charité, Charité - Universitätsmedizin Berlin
This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).
Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach
The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.
A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.
1Department of Diagnostic Radiology, Yale University School of Medicine, 2Department of Psychiatry, Yale University School of Medicine, 3Yale Child Study Center, Yale University School of Medicine, 4Interdepartmental Neuroscience Program, Yale University School of Medicine
Here we present a method for training people to control a brain area involved in contamination anxiety and for probing the relationship between contamination anxiety and brain connectivity patterns.
In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.