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Duke-NUS Medical School

14 ARTICLES PUBLISHED IN JoVE

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Biology

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture
Qianfan Bai *1, Zhiqiang Bai *1, Shaohai Xu 3, Lei Sun 1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, 2Institute of Molecular and Cell Biology, Singapore, 3Division of Bioengineering, School of Chemical & Biomedical Engineering, Nanyang Technological University

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

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Genetics

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
Muhammad Khairul Ramlee 1, Jing Wang 1, Alice M. S. Cheung 1, Shang Li 1
1Cancer & Stem Cell Biology Programme, Duke-NUS Medical School

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

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Immunology and Infection

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva
Tanima Bose 1, Aihua Hou 2, Ryan Lee 2, Louis Tong 2,3,4,5, K. George Chandy 1
1Lee Kong Chian School of Medicine, Nanyang Technological University, 2Singapore Eye Research Institute, 3Singapore National Eye Center, 4Duke-NUS Medical School, 5Yong Loo Lin School of Medicine

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

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Immunology and Infection

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue
Aihua Hou 1,2, Tanima Bose 3, K. George Chandy 3, Louis Tong 1,2,4,5
1Singapore Eye Research Insitute, A Member of SingHealth, 2DUKE-National University of Singapore Medical School, 3Lee Kong Chian School of Medicine, Nanyang Technological University, 4Singapore National Eye Center, 5Yong Loo Lin School of Medicine, National University of Singapore

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

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Neuroscience

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method
Thomas R. Groves 1,2,3, Jing Wang 1,2, Marjan Boerma 1,2, Antiño R. Allen 1,2,3
1Division of Radiation Health, University of Arkansas for Medical Sciences, 2Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 3Neurobiology & Developmental Sciences, University of Arkansas for Medical Sciences

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

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Immunology and Infection

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles
Praveen Kumar Balne 1,2, Veonice Bijin AU 3, Louis Tong 2, Arkasubhra Ghosh 4, Mukesh Agrawal 5, John Connolly 3, Rupesh Agrawal 1
1National Healthcare Group Eye Institute, Tan Tock Seng Hospital, 2Singapore Eye Research Institute (SERI), 3Institute of Molecular and Cell Biology, A*STAR, 4GROW Research Laboratory, Narayana Nethralaya Foundation, 5Vimta Labs Limited

Analysis of tear film cytokines helps in studying various ocular diseases. Bead based multiplex assays are simple and sensitive and enable the testing of multiple targets in samples with small volumes. Here we describe a protocol for tear film cytokine profiling a using bead based multiplex assay.

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Biology

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Xiaofeng Zheng 1,2, Calvin Qing Wei Ho 1, Xiaowei Zheng 3, Kian Leong Lee 4, Katarina Gradin 5, Teresa S. Pereira 3, Per-Olof Berggren 1,2,3, Yusuf Ali 1,2
1Lee Kong Chian School of Medicine, Nanyang Technological University, 2Singapore Eye Research Institute (SERI), Singapore General Hospital, 3The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, 4Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 5Department of Cell and Molecular Biology, Karolinska Institutet

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

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Neuroscience

Intracranial Pharmacotherapy and Pain Assays in Rodents
Erik Martinez 1, Haocheng Zhou 1, Jing Wang 1,2
1Department of Anesthesiology, Perioperative Care and Pain Medicine, New York University School of Medicine, 2Department of Neuroscience and Physiology, New York University School of Medicine

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

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Cancer Research

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
Jiajun Yap 1, Jimin Yuan 1, Zi Heng Tee 1, Xuchao Huang 1, Wan Hwa Ng 1, Jiancheng Hu 1,2
1The Laboratory of Cancer Signaling, Division of Cellular and Molecular Research, National Cancer Centre Singapore, 2The Cancer and Stem Cell Program, Duke-NUS Medical School

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

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Cancer Research

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry
Jenny Lazarus 1, Yagiz Akiska 1, Mirna Perusina Lanfranca 1, Lawrence Delrosario 1, Lei Sun 1, Daniel Long 2, Jiaqi Shi 3, Howard Crawford 2, Marina P. Di Magliano 1, Weiping Zou 1, Timothy Frankel 1
1Department of Surgery, University of Michigan, 2Department of Molecular and Cellular Physiology, University of Michigan, 3Department of Pathology, University of Michigan

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

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Biology

Quantification of Proliferative and Dead Cells in Enteroids
Hua-Shan Li *1, Shao-Fang Xu *1, Jian-Ying Sheng *1, Zhi-Hui Jiang 1, Jing Wang 1, Ning Ding 1, Tao Wang 1, Matthew A. Odenwald 2, Jerrold R. Turner 2,3, Wei-Qi He 1, Hong Xu 1, Juan-Min Zha 1
1Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda (CAM-SU) Genomic Resource Center, Medical College of Soochow University, Department of Oncology, The First Affiliated Hospital of Soochow University, 2Department of Pathology, University of Chicago, 3Department of Pathology, Brigham and Women's Hospital–Harvard Medical School

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

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Medicine

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases
Ivan Seah *1,2, Zengping Liu *1,3,4, Daniel Soo Lin Wong 1, Wendy Wong 2, Graham E. Holder 1,2,5, Veluchamy Amutha Barathi 1,4,6, Gopal Lingam 1,2,4, Xinyi Su 1,2,3,4, Boris V. Stanzel 1,7,8
1Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Ophthalmology, National University Hospital, Singapore, 3Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 4Singapore Eye Research Institute (SERI), 5UCL Institute of Ophthalmology, 6Academic Clinical Program in Ophthalmology, Duke-NUS Medical School, 7Macula Center Saar, Eye Clinic Sulzbach, Knappschaft Hospital Saar, 8Department of Ophthalmology, University of Bonn

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

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Bioengineering

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy
Su Yin Chaw 1,2, Tina Tzee Ling Wong 3,4, Subbu Venkatraman 2,5, Ann-Marie Chacko 1
1Laboratory for Translational and Molecular Imaging, Cancer and Stem Cell Biology Programme, Duke-NUS Medical School, 2School of Materials Science and Engineering, Nanyang Technological University, 3Singapore National Eye Centre, 4Singapore Eye Research Institute, 5Material Science & Engineering, National University of Singapore

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

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Medicine

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice
Sai Bo Bo Tun *1, Edwin Shepherdson *2, Hwee Goon Tay 2,3, Veluchamy Amutha Barathi 1,3,4
1Singapore National Eye Centre, Singapore Eye Research Institute, 2Centre for Vision Research, Duke-NUS Medical School, 3Ophthalmology and Visual Sciences Academic Clinical Program, DUKE-NUS Medical School, 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

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