Jan 13, 2026
Genotyping of variants rs2279238 LXRA and rs2695121 LXRB by qPCR
- Jacqueline Soares Barros Bittar1,
- Nayane Soares de Lima1,
- Caroline Christine Pincela da Costa1,
- 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.
Protocol Citation: Jacqueline Soares Barros Bittar, Nayane Soares de Lima, Caroline Christine Pincela da Costa, Angela Adamski da Silva Reis, Rodrigo da Silva Santos 2026. Genotyping of variants rs2279238 LXRA and rs2695121 LXRB by qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn13xmv5d/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: January 13, 2026
Last Modified: January 13, 2026
Protocol Integer ID: 238499
Keywords: Neurodegenerative diseases, Lipid metabolism, Genetic variants, genotyping of single nucleotide variant, single nucleotide variant, nuclear receptors lxr, activated nuclear receptors lxr, key regulators of lipid metabolism, lipid homeostasi, quantitative pcr, lipid metabolism, variants rs2279238 lxra, rs2695121 lxrb by qpcr lxra, target gene, transcription of target gene, receptor, pcr, qpcr lxra, rs2695121 lxrb, snv, qpcr, cholesterol efflux
Abstract
LXRA and LXRB encode the oxysterol-activated nuclear receptors LXR-alpha and LXR-beta, respectively, which are key regulators of lipid metabolism and neuroprotection. These receptors maintain lipid homeostasis by controlling the transcription of target genes involved in lipid metabolism, storage, transport, and cholesterol efflux - processes that are particularly relevant within the central nervous system. The protocol aims to standardize the genotyping of single nucleotide variants (SNVs) using a quantitative PCR (qPCR) approach.
Materials
- Blood samples (10 mL) with ethylenediaminetetraacetic acid (EDTA)
- Cryotubes
- PureLink® Genomic DNA Kits (ref. K-1820-02)
- Nanodrop® instrument (Thermo Fisher Scientific)
- TaqMan® hydrolysis probes
- QuantStudio 6 Pro Real-time PCR System®
- TaqMan® Genotyping Master Mix® (2X)
- Applied Biosystems™ TaqMan™ SNP Genotyping Assays® (ref. 4351379)
Troubleshooting
Before start
Be sure to wear a coat, mask and gloves. Be careful when manipulating all components of the master mix, preventing contamination.
Before Starting
Be sure to wear a coat, mask and gloves.
Be careful when manipulating all components of the master mix, preventing contamination.
Materials and methods
Study design and ethics
The reliability and validity of study findings depend on well-described and rigorously validated procedures that span the entire research workflow - from ethics approval and definition of the target population to site selection variable specification, materials and methods, data acquisition and analytic tools. In this protocol, genotyping of variants in the two genes under investigation was performed by the quantitative polymerase chain reaction (qPCR) technique. The study was approved by the Ethics and Research Committee of the Federal University of Goiás (CEP/UFG), Brazil, under CAAE number 79593117.7.0000.5083. Participants from both groups were invited and voluntarily agreed to participate in the research and provided written informed consent before enrollment
Variant selection
In this protocol, two SNVs were selected. The first, rs2279238 in NR1H3 (also known as LXRA), is located on chromosome 11p11.2 (GRCh38), exon 4, position 47,260,473. The second, rs2695121 in NR1H2 (LXRB), resides on chromosome 19q13.3 (GRCh38), in the intronic region, at position 50,377,484. The data were extracted from NCBI databases (2023) [1-2] - including dbSNP and Gene - and from relevant literature to inform the study design [3-5]. The methodological workflow is summarized in the flowchart presented in Figure 1; however, this protocol describes only the genotyping procedures.
Figure 1. Diagram illustrating the methodological workflow of genotyping.
The authors, 2026.
Molecular analysis
Genetic analyses were performed on blood samples (10 mL) with ethylenediaminetetraacetic acid (EDTA). Samples were stored in cryotubes at -20 ºC until processing. Genomic DNA was extracted and purified using a silica-based selective DNA-binding method in the presence of chaotropic salts (PureLink‱ Genomic DNA Kits (ref. K-1820-02). DNA concentration was determined by spectrophotometry (1uL) in the Nanodrop‱ instrument (Thermo Fisher Scientific) [6]. TaqMan‱ hydrolysis probes were employed for qPCR on a QuantStudio 6 Pro Real-time PCR System‱ [7]. The qPCR mixture comprised: (i) Genomic DNA at 10ng/uL per reaction; (ii) TaqMan‱ Genotyping Master Mix‱ (2X); and (iii) pre-designed probes from Applied Biosystems‱ TaqMan‱ SNP Genotyping Assays‱ (ref. 4351379), as shown in Table 1.
Table 1. Target sequence corresponding to the TaqMan hydrolysis probe for LXR genes.
Thermo Fisher Scientific‱, 2017 [8].
The probe has two distinct fluorophores: VIC and FAM, responsible for fluorescence when the corresponding allele is identified in the target sequence. One allele is labeled with the fluorescent dye VIC, designed to detect allele 1 (allele C) in the corresponding target sequence, while the second allele is labeled with the dye FAM and targets allele 2 (allele T), as described in Table 2. Both variants involve a single nucleotide substitution, in which cytosine is replaced by thymine (C/T).
Table 2. Hydrolysis probes fluorescence of the alleles involving SNVs rs2279238 LXRA and rs2695121 LXRB.
Thermo Fisher Scientific‱, 2017 [8].
The TaqMan‱ Genotyping Master Mix assay solution is ready to use. Its composition includes ultrapure DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), ROX dye (passive reference), and buffer. To this solution, a specific hydrolysis probe from Applied Biosystems‱ TaqMan‱ SNP Genotyping Assays – ThermoFisher Scientific diluted to a 20x concentration was added. The reagent and probe mixture was prepared for 106 reactions, incorporating a 10% safety margin, as recommended by the manufacturer (96 reactions + 10% margin). A volume of 9µL of the mixture and 1µL of genomic DNA diluted to 10ng/µL was dispensed into each well of the reaction plate, totaling 10µL to fill each of the 96 wells of the optical plate.
Each plate was used to genotype 92 samples from participants in the study, along with two positive controls, one heterozygous (C/T) and one homozygous mutant (T/T), and two negative controls (NTC), which contained only the reagent mixture without the presence of the DNA sample. The reagents were prepared, and the prediluted samples were distributed in a laminar flow hood, following the equipment manufacturer's protocol. After distributing the samples to all wells, the plate was coated with MicroAmp Optical Caps for 96-well plates.
The genotyping assay identifies whether a specific SNP is present or absent by monitoring variations in the fluorescence emitted by the probe-linked dyes. Table 3 outlines the quantities used for the genotyping reaction corresponding to a single assay plate.
Table 3. Description of the quantities used to perform genotyping, referring to a 96-well assay plate.
Thermo Fisher Scientific, 2017 [8].
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.
Table 4. Cycling protocol using validated genotyping assays for qPCR in QuantStudio 6 Pro.
Thermo Fisher Scientific, 2017 [8].
Next, we present the standardized run genotyping workflow of analyses of LXRs variants, in Figure 2.
Figure 2. Standard run for genotyping LXR variants.
Design and Analysis software 2.7.0 da Applied biosystems – Thermofisher, 2026.
Interpretation of results
Genotyping results were analyzed using Diomni‱ Design and Analysis (RUO) in Thermo Fisher Cloud Genotyping application, with data visualized as scatter plots. These plots display genotype clusters, each occupying a specific position and represented by a unique color. Red indicates samples with the wild-type genotype, allele 1 (typically located at the lower right quadrant of the plot. Green represents heterozygous samples, allele 1 and 2, generally positioned in the upper central region. Blue corresponds to mutant genotypes, allele 2, which appear in the upper left quadrant. Positive control samples are denoted by squares in the same color as their respective genotypes. Orange squares indicate negative controls, while orange circles represent samples that failed to amplify. This visualization methodology enhances the interpretation of genotyping results and facilitates the assessment of data quality recorded by the software. The allelic discrimination of the rs2279238 LXRA gene is represented in Figure 3.
Figure 3. Allelic discrimination for genotyping of the genetic variant rs279238, LXRA.
Design and Analysis software 2.7.0 da Applied biosystems – Thermofisher, 2026.
The allelic discrimination of the rs2695121 LXRB genetic variant generated during the amplification process in QuantStudio 6 Pro is shown in Figure 4.
Figure 4. Allelic discrimination for genotyping of the genetic variant rs2695121, LXRB.
Design and Analysis software 2.7.0 da Applied biosystems – Thermofisher, 2026
REFERENCES
[1] NCBIa. NR1H3 Nuclear Receptor subfamily 1 group H member 3 [Homo sapiens (human)] - Gene – NCBI, 2025a. https://www.ncbi.nlm.nih.gov/gene/10062.
[2] NCBIb. NR1H2 Nuclear Receptor Subfamily 1 Group H Member 2 [Homo sapiens 9HUMAN)] - Gene – NCBI. Homo sapiens NR1H2 - Gene – NCBI, 2025b. https://www.ncbi.nlm.nih.gov/gene/?term=Homo+sapiens+NR1H2
[3] Mouzat, K., Molinari, N., Kantar, J., Polge, A., Corcia, P., Couratier, P., Clavelou, P., Juntas-Morales, R., Pageot, N., Lobaccaro, J.-A., Raoul, C., Lumbroso, S., & Camu, W. (2018). Liver X Receptor Genes Variants Modulate ALS Phenotype. Molecular neurobiology, 55(3), 1959–1965. https://doi.org/10.1007/s12035-017-0453-2
[4] Mouzat, K., Chudinova, A., Polge, A., Kantar, J., Camu, W., Raoul, C., & Lumbroso, S. (2019). Regulation of Brain Cholesterol: What Role Do Liver X Receptors Play in Neurodegenerative Diseases? International journal of molecular sciences, 20(16), 3858. https://doi.org/10.3390/ijms20163858
[5] Zhang, H., Lianto, P., Li, W., Xu, M., Moore, J. B., & Thorne, J. L. (2022). Associations between liver X receptor polymorphisms and blood lipids: A systematic review and meta-analysis. Steroids, 185, 109057. https://doi.org/10.1016/j.steroids.2022.109057
[6] 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
[7] 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
[8] 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
Protocol references
[1] NCBIa. NR1H3 Nuclear Receptor subfamily 1 group H member 3 [_Homo sapiens (human)] - Gene – NCBI, 2025a. https://www.ncbi.nlm.nih.gov/gene/10062.
[2] NCBIb. NR1H2 Nuclear Receptor Subfamily 1 Group H Member 2 [_Homo sapiens (human)] - Gene – NCBI. Homo sapiens NR1H2 - Gene – NCBI, 2025b. https://www.ncbi.nlm.nih.gov/gene/?term=Homo+sapiens+NR1H2
[3] Mouzat, K., Molinari, N., Kantar, J., Polge, A., Corcia, P., Couratier, P., Clavelou, P., Juntas-Morales, R., Pageot, N., Lobaccaro, J.-A., Raoul, C., Lumbroso, S., 26 Camu, W. (2018). Liver X Receptor Genes Variants Modulate ALS Phenotype. Molecular neurobiology_, 55(3), 1959–1965. https://doi.org/10.1007/s12035-017-0453-2
[4] Mouzat, K., Chudinova, A., Polge, A., Kantar, J., Camu, W., Raoul, C., 26 Lumbroso, S. (2019). Regulation of Brain Cholesterol: What Role Do Liver X Receptors Play in Neurodegenerative Diseases? International journal of molecular sciences_, 20(16), 3858. https://doi.org/10.3390/ijms20163858
[5] Zhang, H., Lianto, P., Li, W., Xu, M., Moore, J. B., 26 Thorne, J. L. (2022). Associations between liver X receptor polymorphisms and blood lipids: A systematic review and meta-analysis. Steroids_, 185, 109057. https://doi.org/10.1016/j.steroids.2022.109057
[6] 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
[7] 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
[8] 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
Jacqueline Soares Barros Bittar - https://orcid.org/0009-0009-5914-3034
Nayane Soares de Lima - https://orcid.org/0000-0002-5585-6768
Caroline Christine Pincela da Costa - https://orcid.org/0000-0002-6732-913X
Angela Adamski da Silva Reis - https://orcid.org/0000-0002-8281-7334
Rodrigo da Silva Santos - https://orcid.org/0000-0002-9480-4362