Other special issues

Protocols for Coronavirus/COVID-19 Research

With the spread of the COVID-19 (SARS-CoV-2) pandemic across the world, sharing accurate and reproducible methods that can be readily available to the biomedical community became crucial. Therefore, Bio-protocol decided to dedicate a special issue to protocols used in Coronavirus/COVID-19 research in May 2020. By November 2021, we have published 19 high quality protocols used in basic science and clinical research, including diagnostic methods for COVID-19. Additionally, 9 protocols published before May 2020, used in tangential research on other coronaviruses, are also included in this special issue.

More new protocols will be welcomed to be included in this special issue, and we hope it will become a comprehensive resource for detailed protocols relevant to Coronavirus/COVID-19 research (submit here). Typically, we only accept protocols previously used in published articles reporting original research. However, due to the rapid rate of preprint publications during the pandemic, we also include protocols used in preprint articles and preprint versions of the protocols.

Protocols for basic science research

RNA Isolation and Northern Blot Analysis
Authors:  Ying Liao, To Sing Fung, Mei Huang, Shouguo Fang, Yanxin Zhong and Dingxiang Liu, date: 03/20/2014, view: 29291, Q&A: 0

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we examine ATF3, ATF4, and GADD153 gene expression ...

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Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes
Authors:  Amaresh C Panda, Jennifer L. Martindale and Myriam Gorospe, date: 12/20/2016, view: 20642, Q&A: 0

RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al ...

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Measurement of CD8 and CD4 T Cell Responses in Mouse Lungs
Authors:  Craig Fett, Jincun Zhao and Stanley Perlman, date: 03/20/2014, view: 14219, Q&A: 0

Study of the adaptive immune response to a viral challenge in an animal model often includes analysis of the T cell response. Here we discuss in detail the methods that are used to characterize the CD8 and CD4 T cell response following viral challenge in the lung.

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RNA-Affinity Chromatography
Authors:  Lucia Morales , Pedro A. Mateos-Gomez, Luis Enjuanes and Isabel Sola, date: 07/05/2013, view: 12985, Q&A: 0

RNA-affinity chromatography assays are used to identify proteins binding specific RNA sequences. These proteins represent potential factors contributing to the function of RNA molecules. In our lab, we have used this protocol to identify proteins binding sequence motifs involved in replication and transcription of positive strand RNA viruses. The ...

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An Optimized Method for the Production Using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes
Authors:  George Carnell, Keith Grehan, Francesca Ferrara, Eleonora Molesti and Nigel Temperton, date: 08/20/2017, view: 12697, Q&A: 1

The protocol outlined represents a cost-effective, rapid and reliable method for the generation of high-titre viral pseudotype particles with the wild-type SARS-CoV spike protein on a lentiviral vector core using the widely available transfection reagent PEI. This protocol is optimized for transfection in 6-well plates; however it can be readily ...

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Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection
Authors:  Jean Kaoru Millet and Gary R. Whittaker, date: 12/05/2016, view: 11422, Q&A: 0

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway ...

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Virus Infection and Titration of SARS-CoV in Mouse Lung
Authors:  Craig Fett, Jincun Zhao and Stanley Perlman, date: 03/20/2014, view: 11413, Q&A: 0

Two critical steps when investigating an animal model of a virus infection are consistently successfully infecting animals and accurately determining viral titers in tissue throughout the course of infection. Here we discuss in detail how to infect mice with SARS-CoV and then quantify the titer of virus in the lung.

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Simultaneous Intranasal/Intravascular Antibody Labeling of CD4+ T Cells in Mouse Lungs

CD4+ T cell responses have been shown to be protective in many respiratory virus infections. In the respiratory tract, CD4+ T cells include cells in the airway and parenchyma and cells adhering to the pulmonary vasculature. Here we discuss in detail the methods that are useful for characterizing CD4+ T cells in ...

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Biochemical Assays for MTase Activity
Authors:  Yu Chen and Deyin Guo, date: 01/20/2014, view: 8199, Q&A: 0

Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is ...

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Production of the Receptor-binding Domain of the Viral Spike Proteins from 2003 and 2019 SARS CoVs and the Four Common Human Coronaviruses for Serologic Assays and Inhibitor Screening

The recombinant receptor-binding domain (RBD) of the viral spike protein from SARS-CoV-1 and 2 are reliable antigens for detecting viral-specific antibodies in humans. We and others have shown that the levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients are correlated. Here, we report the expression and purification

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Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome

Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses.

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Immunophenotyping and Intracellular Staining of Fixed Whole Blood for Mass Cytometry (CyTOF)
Authors:  Sangeeta Kowli and Holden Maecker, date: 09/05/2020, view: 3648, Q&A: 0

In this report, we present the implementation of mass cytometry for intracellular staining using fixed whole blood. In our assay described here, 250 µl of whole blood, is stimulated in vitro with PMA/ionomycin (or left unstimulated), in the presence of secretion inhibitors (brefeldin A and monensin), lysed-fixed using SMART TUBE buffers, ...

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Computational Analysis and Phylogenetic Clustering of SARS-CoV-2 Genomes
Authors:  Bani Jolly and Vinod Scaria, date: 04/20/2021, view: 3455, Q&A: 1

COVID-19, the disease caused by the novel SARS-CoV-2 coronavirus, originated as an isolated outbreak in the Hubei province of China but soon created a global pandemic and is now a major threat to healthcare systems worldwide. Following the rapid human-to-human transmission of the infection, institutes around the world have made efforts to generate

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Heterologous Expression and Purification of SARS-CoV2 Nucleocapsid Protein
Authors:  Ankur Garg, Lihong Liu, David D. Ho and Leemor Joshua-Tor, date: 08/05/2020, view: 3391, Q&A: 0

This protocol describes a step by step method for heterologous expression of SARS-CoV2 Nucleocapsid (N) protein in Escherichia coli. Moreover, this protocol includes steps to purify the N protein to high purity and homogeneity. Thus, purified protein can be used for ligand binding assays and other biochemical experiments.

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Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome
Authors:  Kashif Aziz Khan and Peter Cheung, date: 12/20/2020, view: 2601, Q&A: 1

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it ...

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Protein Structure Analysis Method of SARS-COV-2 M Protein for Possible Clues Regarding Virion Stability, Longevity and Transmission
Authors:  Saam Hasan and Muhammad Maqsud Hossain, date: 05/19/2020, view: 2128, Q&A: 0

The Severe Acute Respiratory Syndrome Coronavirus 2 or SARS-COV-2 has been the cause of a global pandemic in 2020. With the numbers infected rising well above a 1.9 million and confirmed deaths above 122,000 as of 15th April 2020, it has become the paramount health concern for the global community at present. The SARS-COV-2 genome has ...

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Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes

This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and

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Production of Recombinant Replication-defective Lentiviruses Bearing the SARS-CoV or SARS-CoV-2 Attachment Spike Glycoprotein and Their Application in Receptor Tropism and Neutralisation Assays
Authors:  Nazia Thakur, Giulia Gallo, Ahmed M. E. Elreafey and Dalan Bailey, date: 11/05/2021, view: 1027, Q&A: 0

For enveloped viruses, such as SARS-CoV-2, transmission relies on the binding of viral glycoproteins to cellular receptors. Conventionally, this process is recapitulated in the lab by infection of cells with isolated live virus. However, such studies can be restricted due to the availability of high quantities of replication-competent virus,

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Optimised Method for the Production and Titration of Lentiviral Vectors Pseudotyped with the SARS-CoV-2 Spike

The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike

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A Nucleocapsid-based Transcomplementation Cell Culture System of SARS-CoV-2 to Recapitulate the Complete Viral Life Cycle
Authors:  Yanying Yu, Xiaohui Ju and Qiang Ding, date: 11/05/2021, view: 779, Q&A: 0

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production

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A Protocol for Safe-handling of Remains and Wastes of COVID–19 Patients

The ongoing coronavirus disease-2019 (COVID-19) pandemic has raised significant public health issues which need to be attended. To ensure the containment of various aspects of the pandemic, standard operative procedures (SOPs) are required to enable efficient handling of the situation by the healthcare professionals. Emerging evidence suggest high ...

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Protocols for clinical research

General

Identification and Quantitation of Neutrophil Extracellular Traps in Human Tissue Sections
Authors:  Coraline Radermecker, Alexandre Hego, Philippe Delvenne and Thomas Marichal, date: 09/20/2021, view: 1427, Q&A: 0

Neutrophils are one of the first innate immune cells recruited to tissues during inflammation. An important function of neutrophils relies on their ability to release extracellular structures, known as Neutrophil Extracellular Traps or NETs, into their environment. Detecting such NETs in humans has often proven challenging for both biological

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A Standard Operative Procedure for Safe-handling of Remains and Wastes of COVID-19 Patients

The ongoing coronavirus disease-2019 (COVID-19) pandemic has raised significant public health issues which need to be attended. To enable efficient handling of the situation and prevent the spread of the epidemic, healthcare professionals require various standard operating procedures (SOPs). Emerging evidence suggests high infectivity of the novel ...

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Diagnostic methods for COVID-19 and SARS-CoV-2

COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR

The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and requires a lot of resources. Sample pooling methods improve the efficiency of mass screening and consume

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Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples

During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric

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Colorimetric RT-LAMP Methods to Detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Standard diagnostic methods of Coronavirus Disease 2019 (COVID-19) rely on RT-qPCR technique which have limited point-of-care test (POCT) potential due to necessity of dedicated equipment and specialized personnel. LAMP, an isothermal nucleic acid amplification test (NAAT), is a promising technique that may substitute RT-qPCR for POCT of genomic ...

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A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP)

SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown ...

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Detection of the SARS-CoV-2 Nucleocaspid Protein (NP) Using Immunohistochemistry

While lymphocytopenia is a common characteristic of patients infected by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the mechanisms responsible for this depletion are unclear. With the tissue samples of the spleens and lymph nodes (LNs) from six cases, immunohistochemistry demonstrated ACE2 (angiotensin-converting ...

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A One-enzyme RT-qPCR Assay for SARS-CoV-2, and Procedures for Reagent Production
Authors:  Sanchita Bhadra, Andre C. Maranhao, Inyup Paik and Andrew D. Ellington, date: 01/20/2021, view: 2025, Q&A: 1

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson

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Protocols in progress

VirScan In progress
Contributed by the authors of the following research article:

Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity. Shrock E, Fujimura E, Kula T, Timms RT, Lee IH, Leng Y, Robinson ML, Sie BM, Li MZ, Chen Y, Logue J, Zuiani A, McCulloch D, Lelis FJN, Henson S, Monaco DR, Travers M, Habibi S, Clarke WA, Caturegli P, Laeyendecker O, Piechocka-Trocha A, Li JZ, Khatri A, Chu HY; MGH COVID-19 Collection & Processing Team, Villani AC, Kays K, Goldberg MB, Hacohen N, Filbin MR, Yu XG, Walker BD, Wesemann DR, Larman HB, Lederer JA, Elledge SJ. Science. DOI: 10.1126/science.abd4250

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