Standardization of the amplification of a coding region of APOL1 by RT-PCR in human cell lines.
DOI:
https://doi.org/10.62954/zm7bdn22Keywords:
APOL1, Chronic Kidney Disease, Risk Variants, RT-PCRAbstract
Chronic kidney disease (CKD) represents a growing public health concern in Mexico, where its etiology involves metabolic, environmental, and genetic factors. Among the latter, variants of the APOL1 gene have been associated with an increased risk of CKD in populations of African ancestry; however, their characterization in Latin American populations remains limited. The aim of this study was to standardize an mRNA-based molecular strategy for the amplification of a coding region of APOL1 containing the G1 and G2 risk variants and its characterization by Sanger sequencing. A pair of oligonucleotide primers targeting a coding region within exon 7 of APOL1, corresponding to protein domain IV, was designed. Amplification conditions were optimized by RT-PCR using total RNA extracted from four human cell lines (HaCaT, HeLa, SW-480, and C33A), which were employed as biological models for technical standardization. The resulting amplicons (203 bp) were analyzed by Sanger sequencing to verify the identity of the amplified region.
Reproducible amplification of the expected 203-bp fragment was achieved in all evaluated cell lines. Sanger sequencing confirmed the identity of the amplified product, while complementary bioinformatic analyses (BLASTN, Primer-BLAST, In-Silico PCR, and dbSNP) supported the in silico assessment of primer specificity for the target region of APOL1. No nucleotide changes corresponding to the G1 and G2 risk variants were identified in the analyzed sequences, a finding consistent with the biological model used.
This methodology constitutes a preliminary analytical approach for the amplification and molecular characterization of an APOL1 region harboring the G1/G2 risk variants and provides a methodological basis for future studies aimed at completing the analytical validation of the method through the incorporation of positive controls and previously characterized clinical samples.
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Copyright (c) 2026 Susana A. Rodríguez Jiménez, Gerardo Santos López, Oscar Castañeda Sánchez, Amado I. Grandes Blanco, Lilia Sánchez Minutti, Noe Velázquez Márquez, Dalia Molina Romero, Lorena Milflores Flores, Daniela Rodríguez Flores, Tania Estrada Jiménez, Ericka V. Limón Moreno, Luis Márquez Domínguez (Autor/a)
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