Other special issues

Coronavirus/COVID-19 Special Issue in progress

As the coronavirus pandemic is spreading across the world, now more than ever it is important to promote accurate and reproducible methods and protocols, which are readily available to the bio-medical community. Therefore, Bio-protocol has decided to dedicate a special issue for COVID-19 and other coronavirus-related protocols. We welcome protocols used in basic science research, in medical research, and in bioinformatics. We also welcome protocols from related research on other coronaviruses. We have a dedicated team of editors who will scan the relevant literature and invite authors to submit their protocols. We also welcome authors to send us a presubmission inquiry about their protocols.

Click here to learn more detail about the call for papers on this special issue.

Protocol List

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: 23846, 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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Measurement of CD8 and CD4 T Cell Responses in Mouse Lungs
Authors:  Craig Fett, Jincun Zhao and Stanley Perlman, date: 03/20/2014, view: 11688, 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: 10463, 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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Virus Infection and Titration of SARS-CoV in Mouse Lung
Authors:  Craig Fett, Jincun Zhao and Stanley Perlman, date: 03/20/2014, view: 9354, 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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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: 8491, Q&A: 0

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: 8012, 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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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: 6273, 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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Heterologous Expression and Purification of SARS-CoV2 Nucleocapsid Protein
Authors:  Ankur Garg, Lihong Liu, David D. Ho and Leemor Joshua-Tor, date: 04/29/2020, view: 469, Q&A: 0

This protocol describes 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 (1).

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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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Intracellular Staining on Fixed Whole Blood for Mass Cytometry (CyTOF)
Authors:  Sangeeta Kowli and Holden Maecker, date: 04/23/2020, view: 247, 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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Protein Structure Analysis Method of SARS-COV-2 M Protein for Possible Clues Regarding Virion Stability, Longevity and
Authors:  Saam Hasan and Muhammad Maqsud Hossain, date: 05/19/2020, view: 125, 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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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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COVID-19 detection kits

Extraction, Purification and Detection of SARS-CoV-2 Nucleic Acid
Author:  Shanghai BioGerm Medical Technology Co. LTD, date: 06/20/2020, view: 278, Q&A: 0

Nucleic acid extraction and purification kit is used for isolating high-quality pathogen nucleic acids (DNA/RNA) from a variety of specimens. Nucleic acid can be directly extracted from liquid samples such as blood, serum, plasma, urine, nasopharyngeal swabs, and cell culture. And soil samples such as stool, tissue samples need to be pre- treated, ...

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Protocols in progress

Surveillance of viral shedding (respiratory and fecal) patterns in COVID-19 patients
Contributed by the authors of the following research article:

Prolonged presence of SARS-CoV-2 viral RNA in faecal samples. Wu, Y., Guo, C., Tang, L., Hong, Z., Zhou, J., Dong, X., Yin, H., Xiao, Q., Tang, Y., Qu, X., Kuang, L., Fang, X., Mishra, N., Lu, J., Shan, H., Jiang, G. and Huang, X. The Lancet. DOI: 10.1016/S2468-1253(20)30083-2

Extraction of nucleic acids from the respiratory and fecal samples for 2019-nCoV real-time PCR kit (LifeRiver Ltd. )
Contributed by the authors of the following research article:

Prolonged presence of SARS-CoV-2 viral RNA in faecal samples. Wu, Y., Guo, C., Tang, L., Hong, Z., Zhou, J., Dong, X., Yin, H., Xiao, Q., Tang, Y., Qu, X., Kuang, L., Fang, X., Mishra, N., Lu, J., Shan, H., Jiang, G. and Huang, X. The Lancet. DOI: 10.1016/S2468-1253(20)30083-2

Virus isolation, cell infection, electron microscopy and neutralization assay
Contributed by the authors of the following research article:

A pneumonia outbreak associated with a new coronavirus of probable bat origin. Zhou, P., Yang, X. L., Wang, X. G., Hu, B., Zhang, L., Zhang, W., Si, H. R., Zhu, Y., Li, B., Huang, C. L., Chen, H. D., Chen, J., Luo, Y., Guo, H., Jiang, R. D., Liu, M. Q., Chen, Y., Shen, X. R., Wang, X., Zheng, X. S., Zhao, K., Chen, Q. J., Deng, F., Liu, L. L., Yan, B., Zhan, F. X., Wang, Y. Y., Xiao, G. F. and Shi, Z. L. Nature. DOI: 10.1038/s41586-020-2012-7

Examination of ACE2 receptor for 2019-nCoV infection
Contributed by the authors of the following research article:

Rapid colorimetric detection of COVID-19 coronavirus using a reverse tran-scriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic plat-form: iLACO. Yu, L., Wu, S., Hao, X., Li, X., Liu, X., Ye, S., Han, H., Dong, X., Li, X., Li, J., Liu, J., Liu, N., Zhang, W., Pelechano, V., Chen, W. H. and Yin, X. medRxiv. DOI: 10.1101/2020.02.20.20025874

A dual fluorescent isothermal platform for COVID-19
Contributed by the authors of the following research article:

Rapid colorimetric detection of COVID-19 coronavirus using a reverse tran-scriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic plat-form: iLACO. Yu, L., Wu, S., Hao, X., Li, X., Liu, X., Ye, S., Han, H., Dong, X., Li, X., Li, J., Liu, J., Liu, N., Zhang, W., Pelechano, V., Chen, W. H. and Yin, X. medRxiv. DOI: 10.1101/2020.02.20.20025874

Analysis of a broad spectrum antiviral, β-D-N4-hydroxycytidine, induced-transition mutations and errors rates in coronavirus genome using Primer ID Next-Generation Sequencing
Contributed by the authors of the following research article:

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Sheahan, T. P., Sims, A. C., Zhou, S., Graham, R. L., Pruijssers, A. J., Agostini, M. L., Leist, S. R., Schafer, A., Dinnon, K. H., 3rd, Stevens, L. J., Chappell, J. D., Lu, X., Hughes, T. M., George, A. S., Hill, C. S., Montgomery, S. A., Brown, A. J., Bluemling, G. R., Natchus, M. G., Saindane, M., Kolykhalov, A. A., Painter, G., Harcourt, J., Tamin, A., Thornburg, N. J., Swanstrom, R., Denison, M. R. and Baric, R. S. Sci Transl Med.. DOI: 10.1126/scitranslmed.abb5883

RT-LAMP detection of SARS-CoV-2:primer design and rapid processing of results
Contributed by the authors of the following research article:

Development of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2. Park, G. S., Ku, K., Baek, S. H., Kim, S. J., Kim, S. I., Kim, B. T. and Maeng, J. S. J Mol Diagn.. DOI: 10.1101/2020.03.09.983064

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