May 21, 2026
Taqman SNP Genotyping Assay Protocol: Genotyping of variants rs10139154 and rs61754285 in SCFD1 gene by qPCR
- Caroline Christine Pincela da Costa1,
- Nayane Soares de Lima1,
- Júlia Bispo Campos1,
- Angela Adamski da Silva Reis1,2,
- Rodrigo da Silva Santos1,2,
- Caroline Christine Pincela da Costa3
- 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.;
- 3Universidade Federal de Goiás (UFG)
- Molecular Pathology Laboratory
Protocol Citation: Caroline Christine Pincela da Costa, Nayane Soares de Lima, Júlia Bispo Campos, Angela Adamski da Silva Reis, Rodrigo da Silva Santos, Caroline Christine Pincela da Costa 2026. Taqman SNP Genotyping Assay Protocol: Genotyping of variants rs10139154 and rs61754285 in SCFD1 gene by qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1rex2lr2/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: 317721
Keywords: rs61754285 polymorphisms in the scfd1 gene, rs61754285 in scfd1 gene, significant genetic markers in neurodegenerative research, scfd1 gene, correlation with neurodegenerative phenotype, risk factor for amyotrophic lateral sclerosis, neurodegenerative phenotype, amyotrophic lateral sclerosis, genotyping of variants rs10139154, genetic susceptibility to protein, genotyping of the rs10139154, rs61754285 polymorphism, protein trafficking between the endoplasmic reticulum, significant genetic marker, genomic association study, contribution to motor neuron vulnerability, protein trafficking, neurodegenerative research, resolution results for genomic association study, rs10139154 variant, genetic susceptibility, motor neuron vulnerability, rs61754285 variant, endoplasmic reticulum, trafficking dysregulation, rs10139154, variants rs10139154, rs61754285, essential vesicular transport, gene expression, influencing gene expression, sec1 family domain
Abstract
This protocol describes the genotyping of the rs10139154 and rs61754285 polymorphisms in the SCFD1 gene, which are recognized as significant genetic markers in neurodegenerative research. The SCFD1 gene encodes the Sec1 family domain containing 1 protein, involved in essential vesicular transport and protein trafficking between the endoplasmic reticulum and the Golgi apparatus. The rs10139154 variant has been consistently identified in genome-wide association studies (GWAS) as a risk factor for Amyotrophic Lateral Sclerosis (ALS), potentially influencing gene expression or splicing. Similarly, the rs61754285 variant is investigated for its role in cellular homeostasis and its contribution to motor neuron vulnerability. The joint analysis of these variants aims to characterize genetic susceptibility to protein trafficking dysregulation and its correlation with neurodegenerative phenotypes. The technical focus of this protocol is the standardization of qPCR-based allelic discrimination to ensure high-resolution results for genomic association studies.
Image Attribution
Thermo Fisher Scientific®, 2017 [3].
Materials
- PureLink® Genomic DNA Kit
- NanoDrop™ spectrophotometer
- QuantStudio 6 Pro Real-Time PCR platform
- TaqMan® Genotyping Master Mix
- Applied Biosystems™ SNP Genotyping Assays
- 96-well optical plates
- EDTA-containing tubes
- Cryovials
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, 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.
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.
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 rs10139154 variant is characterized by a C/T transition (adenine/guanine on the opposite strand), and the rs61754285 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.
Thermo Fisher Scientific, 2017 [3].
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 failing to amplify are marked as orange circles. This systematic visual mapping facilitates a robust assessment of genotyping accuracy and overall assay quality. The allelic discrimination of rs10139154 and rs61754285 variants in the SCFD1 gene is represented in Figure 3A and 3B, respectively.
Protocol references
Thermo Fisher Scientific®, 2017 [3].
[1] Thermo Fisher Scientific. (2024). NanoDrop One User Guide. ThermoFisher Scientific. Revision Edition I, March 2023. Willmington: USA.
[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-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
https://assets.thermofisher.com/TFS-Assets/MSD/manuals/nanodrop-one-c-user-guide-EN_20211102.pdf