May 21, 2026
Genotyping of variants rs3774959 in NFKB1 gene and rs4586 in CCL2 gene by qPCR
- Caroline Christine Pincela da Costa1,
- Nayane Soares de Lima1,
- Angela Adamski da Silva Reis2,
- Rodrigo da Silva Santos2
- 1Neurogenetics Research Center, Institute of Biological Sciences (ICB II), Federal University of Goiás (UFG), Goiânia, Goiás, Brazil.;
- 2Department of Biochemistry and Molecular Biology, Institute of Biological Sciences (ICB II), Federal University of Goiás (UFG), Goiânia, Goiás, Brazil.
- Molecular Pathology Laboratory
Protocol Citation: Caroline Christine Pincela da Costa, Nayane Soares de Lima, Angela Adamski da Silva Reis, Rodrigo da Silva Santos 2026. Genotyping of variants rs3774959 in NFKB1 gene and rs4586 in CCL2 gene by qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyom2egx9/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 21, 2026
Last Modified: May 21, 2026
Protocol Integer ID: 317723
Keywords: variants rs3774959 in nfkb1 gene, rs4586 polymorphism in the ccl2 gene, rs4586 snp in the ccl2 gene, rs4586 in ccl2 gene, nfkb1 gene, κb transcription factor, genotyping of the rs3774959 polymorphism, susceptibility to chronic inflammatory disease, ccl2 gene, human inflammatory cascade, chronic inflammatory disease, rs3774959 polymorphism, rs4586 polymorphism, hyperinflammatory phenotype, affecting monocyte recruitment, identification of genetic susceptibility profile, reproducibility in genomic association study, rs4586 snp, genomic association study, genetic susceptibility profile, rs3774959 variant, monocyte recruitment, immune response, genetic susceptibility, variants rs3774959, magnitude of immune response
Abstract
This protocol describes the genotyping of the rs3774959 polymorphism in the NFKB1 gene and the rs4586 polymorphism in the CCL2 gene, both of which are fundamental in regulating the human inflammatory cascade. The rs3774959 variant is associated with modulating the expression of the NF-κB transcription factor, influencing the magnitude of immune responses and susceptibility to chronic inflammatory diseases. Concurrently, rs4586 SNP in the CCL2 gene acts on the regulation of the MCP-1 chemokine, affecting monocyte recruitment to injured tissues. The joint investigation of these variants allows for the identification of genetic susceptibility profiles to hyperinflammatory phenotypes and adverse clinical outcomes in various pathologies. The technical focus of this protocol lies in the standardization of qPCR-based genotyping to ensure reproducibility in genomic association studies.
Image Attribution
Thermo Fisher Scientific®, 2017 [3].
Materials
- TaqMan® Genotyping Master Mix 2x
- TaqMan® SNP Genotyping Assays 20x
- DNA ultrapure water (RNAse free)
- DNA sample (10ng/ µL)
Before start
Be sure to wear a coat, mask and gloves. Be careful when manipulating all components of the master mix, preventing contamination.
Molecular analysis
Peripheral blood (10 mL) was collected in EDTA tubes and stored at -20 °C in cryotubes for subsequent genetic analysis. Genomic DNA isolation was conducted using a silica-column affinity method under chaotropic conditions, following the PureLink® Genomic DNA Kit protocol.
DNA quality and purity were assessed via spectrophotometric analysis (1 µL) using a NanoDrop™ (Thermo Fisher Scientific) [1].
Genotyping was performed by qPCR on a QuantStudio 6 Pro Real-Time PCR System [2], utilizing TaqMan® hydrolysis probes. Each reaction volume consisted of 10 ng/µL of template DNA, TaqMan® Genotyping Master Mix (2X), and specific Applied Biosystems™ SNP Genotyping Assays, as detailed in Table 1.
Allelic discrimination was achieved using dual-labeled probes containing VIC and FAM fluorophores. The system operates through the release of fluorescence upon the detection of specific alleles within the target sequence. Specifically, the VIC dye is assigned to identify the first allele, whereas the FAM dye is utilized for the detection of the second allele, as systematized in Table 2.
In rs3774959 variant involves a single nucleotide substitution, in which adenine is replaced by guanine [A/G]. On the other hand, rs4586 variant involves a substitution of cytosine by thymine (C/T).
Following the manufacturer's guidelines, the reagent-probe master mix included a 10% overage to account for pipetting loss. For the final reactions, 9 µL of this master mix was combined with 1 µL of genomic DNA (standardized at 10 ng/µL) per well. This 10 µL total reaction volume was then dispensed into each well of a 96-well optical plate for processing. For each plate, it was used 92 samples, positive controls (heterozygous and mutant), and two negative controls (NTC). Description for one reaction is on table 3.
The cycling protocol used followed the standard conditions provided by the manufacturer of the TaqMan® hydrolysis probes, as detailed in Table 4. In the plate software, select the Standard mode thermal cycling setting. Figure 2 shows the standardized run genotyping workflow.
Table 3. Description of the quantities used to perform genotyping.
Interpretation of results
Genotype identification was performed using the Thermo Fisher Cloud Genotyping application via Diomni™ Design and Analysis (RUO) software. The results were interpreted through scatter plots, where distinct clusters characterize the different allelic combinations. Specifically, the wild-type genotype (allele 1) is represented by red clusters in the lower right quadrant, while the mutant genotype (allele 2) is shown in blue in the upper left quadrant. Heterozygous samples, containing both alleles 1 and 2, are grouped in green in the upper central area of the plot. Quality control was maintained by including positive controls, marked as squares in the color of the corresponding genotype, and negative controls, indicated by orange squares. Samples that did not undergo amplification are represented by orange circles. This graphical approach ensures a precise evaluation of the genotyping data and software-recorded quality metrics. The allelic discrimination of rs3774959 in the NFKB1 gene and rs4586 in the CCL2 gene is represented in Figure 3A and 3B, respectively.
Figure 3A. Allelic discrimination for genotyping of the genetic variant rs3774959.
Figure 3B. Allelic discrimination for genotyping of the genetic variant rs4586.
Allelic Discrimination Plot
Protocol references
[1] Thermo Fisher Scientific. (2024). NanoDrop One User Guide. ThermoFisher Scientific. Revision Edition I, March 2023. Willmington: USA. https://assets.thermofisher.com/TFS-Assets/MSD/manuals/nanodrop-one-c-user-guide-EN_20211102.pdf
[2] Thermo Fisher Scientific (2025). QuantStudio 6 and 7 Pro Real-Time PCR Systems. https://www.thermofisher.com/br/en/home/life-science/pcr/real-time-pcr/real-time-pcr-instruments/quantstudio-systems/models/quantstudio-6-7-pro.html
[3] Thermo Fisher Scientific. (2017). TaqMan® SNP Genotyping Assays. User Guide. Publication Number MAN0009593. Revision B.0, Life Technologies Corporation | Carlsbad, CA 92008 USA, 2017.https://assets.thermofisher.com/TFS Assets/LSG/manuals/MAN0009593_TaqManSNP_UG.pdf
Acknowledgements
**ORCID**
Caroline Christine Pincela da Costa - https://orcid.org/0000-0002-6732-913X
Nayane Soares de Lima - https://orcid.org/0000-0002-5585-6768
Angela Adamski da Silva Reis - https://orcid.org/0000-0002-8281-7334
Rodrigo da Silva Santos - https://orcid.org/0000-0002-9480-4362