EXPERIMENTAL VALIDATION OF BIOINFORMATICALLY DESIGNED GAPDH PRIMERS FOR RT-QPCR NORMALIZATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS AND LEUKEMIA CELL LINES
Abstract
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a highly sensitive and reproducible molecular method widely used for quantitative gene expression analysis. Reliable normalization of RT-qPCR data depends on the use of stable internal reference genes and well-validated primers. GAPDH is among the most commonly used housekeeping genes due to its constitutive expression in diverse human tissues and cell types. This study aimed to experimentally validate previously in silico-designed GAPDH primer pairs and assess their applicability as reference tools for RT-qPCR normalization in peripheral blood mononuclear cells (PBMCs) and leukemia cell lines (CCRF-CEM and Jurkat). Amplification analysis confirmed efficient and specific GAPDH detection in all tested samples. Among eight designed primer pairs, primer pair 5 demonstrated superior amplification efficiency, optimal GC content, balanced melting temperatures, absence of self-complementarity, and an appropriate amplicon length of 97 bp. Jurkat and CCRF-CEM cell lines exhibited earlier Ct values compared to PBMCs, reflecting differences in GAPDH expression levels. No amplification was observed in the negative control, confirming assay specificity. The findings indicate that the selected GAPDH primer pair is suitable for accurate RT-qPCR normalization in both normal blood-derived cells and leukemia models.
Keywords
RT-qPCR, GAPDH, housekeeping gene, primer validation, PBMC, leukemia cell lines, CCRF-CEM, Jurkat, gene expression normalization.
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