Jun 06, 2025
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
- Giovanna Ribeiro Sousa de Araujo1,
- Raquel da Silva Carvalho2,
- Vanessa Rafaela Milhomen Cruz Leite3,
- Jessica Barletto de Sousa Barros4,
- Irmtraut Araci Hoffmann Pfrimer4
- 1Undergraduate student in the Biomedical Science program at the Pontifical Catholic University of Goiás (PUC-GO). Member of the Center for Immunological Studies and Research (NEPY);
- 2Ph.D. student in Environmental and Health Sciences (PPGCAS) at the Pontifical Catholic University of Goiás (PUC-GO). Member of the Center for Immunological Studies and Research (NEPY);
- 3Laboratory of Genetics and Molecular Biology - Institute of Biological Sciences II - Federal University of Goiás, Goiânia, Goiás;
- 4Ph.D. in Environmental and Health Sciences (PPGCAS) at the Pontifical Catholic University of Goiás (PUC-GO). Member of the Center for Immunological Studies and Research (NEPY).
- Giovanna Ribeiro Sousa de Araujo: *These authors contributed equally to this work.;
- Raquel da Silva Carvalho: *These authors contributed equally to this work.;
- Jessica Barletto de Sousa Barros: #These authors contributed equally to this work.;
- Irmtraut Araci Hoffmann Pfrimer: #These authors contributed equally to this work.
Protocol Citation: Giovanna Ribeiro Sousa de Araujo, Raquel da Silva Carvalho, Vanessa Rafaela Milhomen Cruz Leite, Jessica Barletto de Sousa Barros, Irmtraut Araci Hoffmann Pfrimer 2025. Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvowzrdl4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 27, 2025
Last Modified: June 06, 2025
Protocol Integer ID: 219016
Keywords: Tumor Necrosis Factor alpha, Dengue, Single Nucleotide Polymorphism, rs361525, SNP -238 G>A, immune response to dengue, dengue virus, single nucleotide polymorphism snp tnf, rs361525 polymorphism in the tnf, control study in the brazilian midwest dengue, arboviral disease of major public health importance, arboviral disease, brazilian midwest dengue, dengue, tumor necrosis factor, susceptibility to infectious disease, promoter region of the tnf, rs361525 polymorphism, allelic frequencies of the rs361525 tnf, infectious disease, rs361525 tnf, associations between genotype, gene in individual, regulation of this cytokine, better understanding of the genetic factor, cytokine, genotype, genetic factor, infection status, immune response
Funders Acknowledgements:
Fundação de Amparo à Pesquisa do Estado de Goiás (Fapeg)
Conselho Nacional de Desenvolvimento Científico (CNPQ)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Abstract
Dengue is an arboviral disease of major public health importance, particularly in tropical regions such as the Brazilian Midwest. The host's inflammatory response plays a key role in the clinical progression of the disease, with tumor necrosis factor-alpha (TNF-α) being one of the central cytokines in this process. The single nucleotide polymorphism SNP TNF-α rs361525, located in the promoter region of the TNF-α gene, has been associated with the regulation of this cytokine's expression and susceptibility to infectious diseases with hepatitis C (HCV) and hepatitis B (HBV). This case-control study aimed to investigate the genotypic and allelic frequencies of the rs361525 TNF-α SNP in individuals infected with the dengue virus compared to healthy controls residing in the Brazilian Midwest region. Genotyping was performed using real-time PCR, and the data were analyzed for associations between genotypes and infection status. The findings may contribute to a better understanding of the genetic factors involved in the immune response to dengue.
Attachments
Materials
- Roll of paper for sanitizing the cooling box during movement and protecting the PCR plate after reading;
- PCR cooling box to place the PCR plate in during handling;
- Notebook and pen for making calculations, notes and relevant observations.
Troubleshooting
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
General aspects
Definition:
Genotyping is a tool in scientific research that allows genetic variability to be studied and associated with specific characteristics. In this way, it participates in genetic tests that make it possible to identify genetic variations in studies focused on single nucleotide polymorphisms (SNPs).
Objectives:
To evaluate the use of qPCR to identify the SNP TNF-α rs361525 in DNA samples in order to guarantee the reliability of the results obtained, providing a standardized methodology for the genetic analysis of the presence of this SNP in individuals with dengue.
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Personal Protective Equipment (PPE)
- Powder-free nitrile gloves;
- Lab coat;
- Closed shoes and pants.
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Materials
- TaqMan probe (Thermo Fisher) of SNP TNF-α rs361525 diluted 1:1 with ultra-pure water (C___2215707_10).
- Roll of paper for sanitizing the cooling box during movement and protecting the PCR plate after reading;
- PCR cooling box to place the PCR plate in during handling;
- Notebook and pen for making calculations, notes and relevant observations.
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Procedure
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Check that the stabilizer is connected to the 220V socket.
Lightcycler 480. Switch on the Lightcycler 480.
Red lights will come on, wait for the green light to appear to turn on the computer.
Switching on the equipmen Lightcycler 480.
Lightcycler 480 on.
Turn on the computer and wait for the start screen to appear.
As soon as the screen below appears, click F1.
Turn on the computer to start studying.
Enter the login and password from the first access.
Open the Lightcycler 480 program.
Lightcycler 480 program.
Enter the login and password for the second access.
Location of login and password to access the program.
Click on “New Experiment from Template”.
Select the following standardization already saved on the computer:
..Check that you are in 4 schedules of 40 cycles each, as below:
At the end, click on “Apply Template”.
PCR cycles.
Click on the side button of the Lightcycler 480 to exit the treadmill.
Carefully remove the PCR plate from the cooling box, touching only the sides, and place it on the conveyor belt.
ATTENTION:
Observe the edge of the diagonal side and be careful not to touch the barcode.
Lightcycler 480 switched on and running.
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Click the button again to close the belt.
At the top, under “Window”, identify the PCR that is being made:
Select the folder.
It is necessary to identify with the type of sample (DNA), number of samples (SAMPLES, CP, CN, BR, TOTAL REACTIONS) and rs361525.
Select “Start Run”.
Then wait for the heating graph to appear.
Wait an average of 40' to 1 hour for the plate to be read.
Record the fluorescence graph.
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
End of PCR.
The photo below shows the end of a qPCR.
Click on “Analysis” on the side of the page.
Click on “Endpoint Genotyping” and “OK”.
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Select the colors VIC and FAM:
VIC (ALEL X): VIC/HEX/Yellow555 (533-580)
FAM (ALEL Y): FAM (465-510)
Place to choose the colors according to the Thermo Fisher protocol.
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Select the wells that have been pipetted and check the filters.
After selecting the wells, click on “Calculate”.
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Analyze the presence of the alleles and write down their respective genotypes in the notebook.
In order to genotype the samples, the following data from the duplicates is required:
ALEL X - GENOTYPE A
ALEL Y - GENOTYPE G
Example of genotyping after calculations:
Thus, when analyzing the samples, they can be considered wild, mutant and heterozygous:
ALEL GENOTYPING
YY-GG WILD
XX-AA MUTANT
XY-GA HETEROZYGUS
Genotyping of the rs361525 polymorphism in the TNF-α gene in individuals infected with the Dengue virus: A case-control study in the Brazilian Midwest
Actions in cases of non-compliance
- Record any complications in the ATA notebook;
- Contact the counselors;
- Make photographic records;
- If there are any problems with the product or equipment, contact technical assistance.
Send the analysis for statistical study
The analysis of the genotypes correlated with the symptoms presented by individuals infected with Dengue by statistical study.
Protocol references
1-LABVITAL. O que é genotipagem? Disponível em: https://labvital.com.br/glossario/o-que-e-genotipagem/#:~:text=A genotipagem é essencial para,e na prevenção de doenças. Acesso em: 14 nov. 2024.
2-SANTOS, N. C. D. et al. Association of single nucleotide polymorphisms in TNF-α (-308G/A and -238G/A) to dengue: Case-control and meta-analysis study. Cytokine, v. 134, p. 155183, Out. 2020. DOI: 10.1016/j.cyto.2020.155183. Epub 27 Jul. 2020. PMID: 32731142.
3-THERMO FISHER SCIENTIFIC. Gene expression analysis & genotyping. Disponível em: https://www.thermofisher.com/br/en/home/life-science/gene-expression-analysis-genotyping/genotyping-genomic-profiling.html. Acesso em: 14 nov. 2024.
4-ZHENG, M. et al. The TNF-α rs361525 and IFN-γ rs2430561 polymorphisms are associated with liver cirrhosis risk: a comprehensive meta-analysis. Frontiers in Immunology, Lausanne, v. 14, p. 1–12, 14 abr. 2023. DOI: 10.3389/fimmu.2023.1129767.
Acknowledgements
Funding:
1- National Council for Scientific and Technological Develompment (CNPQ).
2-Research Support Foundation of the State of Goiás (FAPEG).
3-Coordination for the Improvement of Higher Education Personnel (CAPES).
4-Pontifical Catholic University of Goiás (PUC-GO).
I'm grateful for the support and encouragement of my advisors, Master's Raquel da Silva Carvalho, Dr. Irmtraut Araci Hoffmann Pfrimer and Dr. Jéssica Barletto de Sousa Barros. In particular, my colleagues and friends João Otávio Alves Pereira, Jônatas Ferreira Vilela, Rute da Silva Carvalho and Maike Vieira Sabath who accompanied me throughout this journey, making it lighter and more special.