May 25, 2026
Taqman SNP Genotyping Assay Protocol: Genotyping of variants rs61742937 and rs151117904 in the SETX gene by qPCR
- Caroline Christine Pincela da Costa1,
- Nayane Soares de Lima1,
- Isabela Rodrigues Resende1,
- Angela Adamski da Silva Reis1,2,
- Rodrigo da Silva Santos1,2
- 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, Isabela Rodrigues Resende, Angela Adamski da Silva Reis, Rodrigo da Silva Santos 2026. Taqman SNP Genotyping Assay Protocol: Genotyping of variants rs61742937 and rs151117904 in the SETX gene by qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp7418gzp/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 22, 2026
Last Modified: May 25, 2026
Protocol Integer ID: 317785
Keywords: setx gene encodes senataxin, setx gene by qpcr, significant genetic markers in neurodegenerative research, polymorphisms in the setx gene, taqman snp genotyping assay protocol, setx gene, neurodegenerative phenotype, significant genetic marker, genotyping of variant, mutations in setx, rs151117904 polymorphism, amyotrophic lateral sclerosis, including amyotrophic lateral sclerosis, genetic susceptibility, genetic susceptibility to cellular stress, correlations with neurodegeneration, genomic integrity, maintenance of genomic integrity, standardization of qpcr, neurodegeneration, genomic association study, effective results for genomic association study, mutation, neurodegenerative research, motor neuron vulnerability, rna helicase, dna damage accumulation, rna helicase essential for rna processing, ataxia
Abstract
This protocol describes the genotyping of the rs61742937 and rs151117904 polymorphisms in the SETX gene, which are recognized as significant genetic markers in neurodegenerative research. The SETX gene encodes senataxin, a DNA/RNA helicase essential for RNA processing, transcription termination, and the maintenance of genomic integrity. Variants and mutations in SETX have been consistently linked to motor neuron vulnerability and neurodegenerative phenotypes, including amyotrophic lateral sclerosis (ALS) and ataxia. The investigation of rs61742937 and rs151117904 aims to characterize genetic susceptibility to cellular stress, DNA damage accumulation, and their correlations with neurodegeneration. The technical focus of this protocol is the standardization of qPCR-based allelic discrimination using TaqMan® hydrolysis probes, ensuring high-resolution, reproducible, and cost-effective results for genomic association studies in diverse populations.
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
To facilitate genetic testing, 10 mL of blood was drawn into EDTA-containing tubes and kept at -20 °C in cryovials. The extraction of genomic DNA was performed using the PureLink® Genomic DNA Kit [1], which employs chaotropic salts and silica-based membrane affinity. A NanoDrop™ spectrophotometer (Thermo Fisher Scientific) was used to quantify the isolated DNA and verify purity using 1 µL aliquots. Target SNPs were genotyped via qPCR on the QuantStudio 6 Pro Real-Time PCR platform, employing TaqMan® chemistry for allelic discrimination [2]. Standard reaction mixtures included 10 ng/µL of genomic DNA, 2X TaqMan® Genotyping Master Mix, and pre-designed Applied Biosystems™ SNP Genotyping Assays, with specific details provided in Table 1.
Dual-labeled TaqMan® probes, incorporating FAM and VIC fluorophores, were utilized for allelic differentiation. This detection system relies on the emission of fluorescence triggered by the identification of specific target sequences. As detailed in Table 2, the VIC reporter dye was calibrated to detect the primary allele, while the FAM dye was assigned to the secondary allele. Regarding the specific targets, the rs61742937 variant is characterized by a C/T transition (adenine/guanine), and the rs151117904 variant consists of an A/G substitution (cytosine/thymine). This experimental setup allows for the clear distinction between homozygous and heterozygous genotypes through the ratio of the two fluorescent signals.
The qPCR setup utilized the TaqMan® Genotyping Master Mix, which provides a stabilized environment with ultrapure DNA polymerase, dNTPs, buffer, and ROX for passive reference normalization. This solution was supplemented with 20x Applied Biosystems™ SNP Genotyping Assays (ThermoFisher Scientific). To compensate for volume loss during pipetting, the master mix was prepared with a 10% surplus according to manufacturer instructions. Final reactions were carried out in a 10 µL total volume, consisting of 9 µL of the reagent-probe mixture and 1 µL of genomic DNA template (adjusted to a concentration of 10 ng/µL). The assays were performed in 96-well optical plates, each containing 92 experimental samples, alongside positive controls (representing heterozygous and mutant genotypes) and duplicate non-template controls (NTC). Detailed reaction components are specified in Table 3.
The thermal cycling parameters were set according to the standard protocols established by the manufacturer for TaqMan® hydrolysis assays, with specific stage details provided in Table 4. When configuring the instrument software, the Standard cycling mode was selected for the run. The systematic procedure for the genotyping analysis, from initial denaturation to final signal detection, is visually outlined in the standardized workflow presented in Figure 2.
Figure 2. Standard run for genotyping the rs61742937 and rs151117904 variants in the SETX gene.
Interpretation of results
Data analysis was conducted using the Diomni™ Design and Analysis (RUO) software via the Thermo Fisher Cloud Genotyping platform. Allelic discrimination was determined through scatter plot visualization, where samples are grouped into clusters based on their fluorescence profiles. In this spatial distribution, red clusters located in the lower right quadrant signify the wild-type genotype (allele 1), whereas blue clusters in the upper left quadrant identify the mutant genotype (allele 2). Heterozygous instances, possessing both alleles, are clustered in green within the upper central region. To validate the run, positive controls are highlighted as squares matching their respective genotype colors, while negative controls (NTC) appear as orange squares. Any samples that fail to amplify are marked with orange circles. This systematic visual mapping facilitates a robust assessment of genotyping accuracy and overall assay quality. The allelic discrimination of the rs61742937 and rs151117904 variants in the SETX gene is shown in Figures 3A and 3B, respectively.
Allelic Discrimination Plot
Figure 3A. Allelic discrimination for genotyping of the genetic variant rs61742937.
Protocol references
[1] PureLink® Genomic DNA Kit. [2] QuantStudio 6 Pro Real-Time PCR platform. [3] Thermo Fisher Scientific®, 2017.
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