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Authors: Magda Bletsa, Antonios Fikatas, Yiqiao Li, Sophie Gryseels, Jan Felix Drexler, Philippe Lemey
Summary: This protocol can be used for total RNA purification from tissue samples. It includes an intermediate on-column DNase treatment, which results in higher RNA yield and purity. This method is mainly optimised to obtain hepacivirus RNA extracts for whole-genome sequencing, but it can also be used for any viral RNA purification from samples with lower viral loads.

Proper citation: Magda Bletsa, Antonios Fikatas, Yiqiao Li, Sophie Gryseels, Jan Felix Drexler, Philippe Lemey 2020. High-quality RNA purification with on-column DNase treatment from tissue specimens . protocols.io https://dx.doi.org/10.17504/protocols.io.8ufhwtn Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:Target cells are either selected or depleted by incubating the sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. If these are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.Sample Preparation:Enzymatic digestion of mouse brain followed by myelin removal is recommended to achieve the highest purity and yield. There are several protocols published that can be applied. As a general guideline, Trypsin digestion followed by a 70/37/30% percoll gradient will increase final purity and yield.Scale up volumes if using 14 ml tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Selection Kits Protocol - 5. protocols.io https://dx.doi.org/10.17504/protocols.io.7z9hp96 Copy   

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Authors: Serena Ding
Group: Behavioural Genomics
Summary: For imaging swarming behaviour of 40 young adult C. elegans on agar using the Phoenix multi-worm tracker system. Worms are synchronised by bleaching and refeeding for 72 hours, and then 40 young adult hermaphrodites are transferred by glass pipette onto a 35 mm regular NGM plate for imaging for 7 hours at 25 fps.

Proper citation: Serena Ding 2019. bright field standard swarming imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.vybe7sn Copy   

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Authors: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: SalivaDirect is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets.Version 2 includes: Optimized thermocycler conditionsLocally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclersUse of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates.Version 3 has been updated to remove steps for sample self-collection.Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx.

Proper citation: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie 2020. SalivaDirect: RNA extraction-free SARS-CoV-2 diagnostics. protocols.io https://dx.doi.org/10.17504/protocols.io.bjswknfe Copy   

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Authors: Aadi Varma

Proper citation: Aadi Varma 2017. Testing. protocols.io https://dx.doi.org/10.17504/protocols.io.k8iczue Copy   

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Authors: Pablo Delgado-Galindo, Javier Rodríguez-Santero, Juan Jesus Torres-Gordillo
Summary: La tabla forma parte de una revisión sistemática de investigaciones de impacto del periodo 2014-2019 que muestran los factores contextuales que influyen en la eficacia escolar. El objetivo de conocer estos factores es tener un mayor conocimiento de qué influye en la eficacia escolar para mejorar las prácticas orientadoras. .justify:after { content: ""; display:inline-block; width: 100%; } La tabla recoge las publicaciones ordenadas de mayor a menor factor de impacto en su año de publicación. En el caso de los libros y capítulos de libro, se indica su inclusión en el ranking Scholary Publishers Indicators (SPI). Las tesis doctorales se encuentran en repositorios institucionales de prestigio. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Pablo Delgado-Galindo, Javier Rodríguez-Santero, Juan Jesus Torres-Gordillo 2020. Investigaciones de impacto que estudian los factores contextuales que afectan a la Eficacia Escolar en el periodo 2014-2019. protocols.io https://dx.doi.org/10.17504/protocols.io.bpbnmime Copy   

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Authors: Rabia Khan

Proper citation: Rabia Khan 2016. Mouse BMDM. protocols.io https://dx.doi.org/10.17504/protocols.io.gcqbsvw Copy   

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Authors: Ekaterina Pokrant, Francisca Medina, Aldo Maddaleno, Betty San Martin, Javiera Cornejo
Summary: Analytical methodology for the detection of sulfachloropyridazine (SCP) in samples of feathers via LC-MS/MS was implemented based on techniques previously published by other authors:  1- Hindle R. A validated atmospheric pressure chemical ionization method for analysing sulphonamides in pork muscle. 2013. Available at http://www.youngin.com/application/0411-0045EN-E.pdf. Accessed October 12, 2017. 2- Renew JE, Huang CH. Simultaneous determination of fluoroquinolone, sulfonamide, and trimethoprim antibiotics in wastewater using tandem solid phase extraction and liquid chromatography–electrospray mass spectrometry. J Chromatogr A 2004; 1042: 113-21. 3- Shao B, Dong D, Wu y, Hu J, Meng J, Tu X, Xu S. Simultaneous determination of 17 Sulfonamide residues in porcine meat, kidney and liver by solid phase extraction and liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2005; 546:174-81. 4- Pang G, Cao YZ, Zhang JJ, Jia GQ, Fan CL, Li XM, Liu YM, Li ZY, Shi YQ. Determination of sulfonamides in honey by liquid chromatography- tandem mass spectrometry. J AOAC Int 2005; 88:1304-11. 5- Stubbings G, Bigwood T. The development and validation of a multi-class liquid chromatography tandem mass spectrometry (LC– MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach. Anal Chim Acta 2009; 637:68-78. 6- Bedendo GC, Jardim IC, Carasek E. A simple hollow fiber renewal liquid membrane extraction method for analysis of sulphonamides in honey samples with determination by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010; 1217:6449-54. 7- Yu H, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high-performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulphonamides in the foods of animal origin. J Chromatogr B 2011; 879:2653-62. The method is based mainly on a solid-liquid extraction with organic solvents. The analyte was concentrated using a water bath at 40-50°C under a mild nitrogen flow. For the instrumental analysis, a Symmetry C8 analytical column of 3.5μm and 2.1 x 100mm (Waters®) was fitted in an Agilent 1290 infinity series liquid-chromatograph equipment, coupled to an API 3200 (AB Sciex, Darmstadt, Germany) triple-quadrupole mass-spectrometer. The analytical data was then integrated using the Analyst® version 1.5 software package (SCIEX, Framingham, Massachusetts).

Proper citation: Ekaterina Pokrant, Francisca Medina, Aldo Maddaleno, Betty San Martin, Javiera Cornejo 2018. Chemical extraction of sulfachloropyridazine from muscle samples. protocols.io https://dx.doi.org/10.17504/protocols.io.pvcdn2w Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. Th2 lymphocytes synthesize IL-19 and acts on resident vascular cells in addition to immune cells. It is an anti-inflammatory molecule. It promotes immune responses mediated by regulatory lymphocytes and has substantial activity on microvascular.[1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.

Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of IL-19 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj3jkqkn Copy   

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Authors: Roey Angel, Eva Petrova
Group: SoWa RI Anaerobic and Molecular Microbiology (public)
Summary: Universal 16S rRNA probe-based-qPCR assay for bacteria.The primers target the V4 region of the 16S rRNA gene and were specifically designed for Illumina amplicon sequencing. The original primers were designed by Caporaso et al. (2012) and modified by Walters et al. (2015). For barcoding, we use the Fludigm Access Array for barcoding the sample and therefore the primers are synthisized with the CS1 and CS2 regions.

Proper citation: Roey Angel, Eva Petrova 2020. General bacteria and archaea 16S-rRNA (515Fmod-806Rmod) for Illumina amplicon sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bnf7mbrn Copy   

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Authors: Bonnie Hurwitz
Group: Hurwitz Lab, MetaFunc Course

Proper citation: Bonnie Hurwitz 2016. MG_HW10: Annotate anvio gene calls for function and taxonomy. protocols.io https://dx.doi.org/10.17504/protocols.io.gczbsx6 Copy   

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Authors: Angel Justiz-Vaillant, Suzette E Curtello
Group: University of the West Indies, [email protected]
Summary: The United States veterinarian Daniel E Salmon discovered the genus Salmonella. Salmonella is non-spore forming and it may survive without oxygen. This bacterial strain is mainly associated with animals. Salmonella is ubiquitous and may be asymptomatic which has generated great health concern.This bacterium is associated with life threatening diseases among human and animals.Gastroenteritis is one such disease which may resolve itself within five days in healthy individuals.Immuno compromised individuals and the young are at great risk as this may progress to secondary systemic complication.Salmonella serotype exceeds two thousand which may be differentiated from each other by the International Kauffmann-white Scheme Serotyping Manual.Surface antigens produced by salmonellae are used in the determination of isolated strain.Determination of the “O” antigen group associated with the carbohydrate constituent of the lipopolysaccharide membrane of the bacterium precede the identification of the protein “H” antigen which is linked to the tail like flagella of the bacterium.The two existing phases of Salmonellamay be referred to as specific and nonspecific which function as motile and non motile phases.The existing phase of Salmonellawill determine the “H” antigens that are produced.An additional “Vi” antigen is associated with virulent encapsulated strains of Salmonella Typhi.

Proper citation: Angel Justiz-Vaillant, Suzette E Curtello 2020. Salmonella purification. protocols.io https://dx.doi.org/10.17504/protocols.io.bjjekkje Copy   

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Authors: Angel Justiz-Vaillant
Summary: A protein that combines the binding capacity of SpA and SpG is not comercially available. It can be easy created in the laboratory by combining these two immunoglobulin-binding proteins to horseradish peroxidase by the periodate method [1]. However, a mixture of SpA and SpG could have the same effect as universal reagent in immunodetection. 1. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175

Proper citation: Angel Justiz-Vaillant 2020. Universal Immunoblot analysis for investigating Protein-AG (SpAG)-binding to avian and mammalian immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjseknbe Copy   

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Authors: Bieke Tack
Summary: Standard operating procedures for Salmonella blood culture surveillance of the unit of tropical bacteriology at the Institute of Tropical Medicine in Antwerp (Belgium):- Blood culture work-up- Pathogen identification- Salmonella serotyping- Antibiotic susceptibility testing

Proper citation: Bieke Tack 2019. Salmonella blood culture surveillance: SOP's ITM. protocols.io https://dx.doi.org/10.17504/protocols.io.9rah52e Copy   

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Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Following Day 3 replating, i3Neurons should be cultured for at least 1 week before lysis, fixation, or other experimental endpoint, and they remain viable for at least 1 month with proper maintenance. This protocol covers medium conditions, coating of tissue culture dishes with synthetic polymers, recommended plating densities, and maintenance procedures for long-term culture and for specific experimental applications.Cortical Neuron Culture Medium (CM) is sufficient to promote the maturation and longterm maintenance of i3Neurons in culture. i3Neurons express general markers of cortical neurons, as well as specific pre- and postsynaptic markers of glutamatergic excitatory cortical neurons. Since these cells are post-mitotic after 3 days of differentiation (see Basic Protocol 5) and prefer neuron-conditioned medium to fresh medium, maintenance conditions for these cultures are minimal. Generally, half-medium changes every 7 days with fresh, pre-warmed CM are sufficient for culturing beyond d10 (7 days after replating). Neuron attachment and growth also require a strongly adhesive substrate. Coating plates with synthetic polymers such as poly-L-ornithine (PLO) is necessary for i3Neuron attachment, viability, and successful outgrowth.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. CULTURING i3NEURONS (Basic Protocol 6). protocols.io https://dx.doi.org/10.17504/protocols.io.5jpg4mn Copy   

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Authors: Debora Ferreira Barreto-Vieira, Fernanda Cunha Jácome, Marcos Alexandre Nunes da Silva, Gabriela Cardoso Caldas, Elen Mello de Souza, Audrien Alves Andrade, Ortrud Monika Barth.

Proper citation: Debora Ferreira Barreto-Vieira, Fernanda Cunha Jácome, Marcos Alexandre Nunes da Silva, Gabriela Cardoso Caldas, Elen Mello de Souza, Audrien Alves Andrade, Ortrud Monika Barth. 2017. Cell fixation for processing for analysis by transmission electron microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.jqucmww Copy   

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Authors: Lani Tieu, Lauren Smith, Olivier George
Group: George Lab

Proper citation: Lani Tieu, Lauren Smith, Olivier George 2020. Minipump Subcutaneous Implantation for Rats. protocols.io https:// Copy   

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Authors: Wendkouni Adelphe Sabine OUEDRAOGO, Sandrine Biau-Lalanne, Emmanuel Bonnet, Valery Ridde

Proper citation: Wendkouni Adelphe Sabine OUEDRAOGO, Sandrine Biau-Lalanne, Emmanuel Bonnet, Valery Ridde 2020. Scoping review protocol on the use of telephone in the implementation of citizen participation in the processes of developing health system strengthening reforms/policies in Sub-Saharan Africa (participation in decision-making). protocols.io https://dx.doi.org/10.17504/protocols.io.bky7kxzn Copy   

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Authors: Roey Angel, Eva Petrova, Ana Lara
Group: SoWa RI Anaerobic and Molecular Microbiology (public)
Summary: Universal 16S rRNA probe-based-qPCR assay for bacteria.The primers and probe are taken from Yu et al. (2005).

Proper citation: Roey Angel, Eva Petrova, Ana Lara 2020. qPCR: Bacterial SSU rRNA 338F-516P-805R. protocols.io https://dx.doi.org/10.17504/protocols.io.bqx5mxq6 Copy   

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Authors: Dr Vikas Pandey, Dr Saurabh Singh, Shruti Ahuja, Avishek Dev, Manu Kadyan, Mukesh Pandit, Amir Khan, Sambit Dam, Tanish Garg
Group: XPRIZE Rapid Covid Testing, Valetude Primus Healthcare, New Delhi
Summary: Valetude Primus Healthcare (VPH) is a healthcare spinoff from IIT Delhi, India. VPH has developed an automated portable device, called AutoGene-COVID, to detect SARS COV 2 infection from a patient’s nasopharyngeal, oropharyngeal, nasal swab, or saliva sample within 2 hours. The one-step rapid process includes RNA extraction and purification using magnetic capture (

Proper citation: Dr Vikas Pandey, Dr Saurabh Singh, Shruti Ahuja, Avishek Dev, Manu Kadyan, Mukesh Pandit, Amir Khan, Sambit Dam, Tanish Garg 2020. VPH_AUTOGENE-COVID_Diagnosis_Protocol_XPRIZE. protocols.io https://dx.doi.org/10.17504/protocols.io.bkxjkxkn Copy   

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