Validation of Endogenous Control Genes by Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction for Acute Leukemia Gene Expression Studies.

Genes 2024 Jan 24

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Reference genes are used as internal reaction controls for gene expression analysis, and for this reason, they are considered reliable and must meet several important criteria. In view of the absence of studies regarding the best reference gene for the analysis of acute leukemia patients, a panel of genes commonly used as endogenous controls was selected from the literature for stability analysis: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Abelson murine leukemia viral oncogene human homolog 1 (ABL), Hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Ribosomal protein lateral stalk subunit P0 (RPLP0), β-actin (ACTB) and TATA box binding protein (TBP). The stability of candidate reference genes was analyzed according to three statistical methods of assessment, namely, NormFinder, GeNorm and R software (version 4.0.3). From this study's analysis, it was possible to identify that the endogenous set composed of ACTB, ABL, TBP and RPLP0 demonstrated good performances and stable expressions between the analyzed groups. In addition to that, the GAPDH and HPRT genes could not be classified as good reference genes, considering that they presented a high standard deviation and great variability between groups, indicating low stability. Given these findings, this study suggests the main endogenous gene set for use as a control/reference for the gene expression in peripheral blood and bone marrow samples from patients with acute leukemias is composed of the ACTB, ABL, TBP and RPLP0 genes. Researchers may choose two to three of these housekeeping genes to perform data normalization.

Citation

Flávia Melo Cunha de Pinho Pessoa, Vitória Beatriz de Jesus Viana, Marcelo Braga de Oliveira, Beatriz Maria Dias Nogueira, Rodrigo Monteiro Ribeiro, Deivide de Sousa Oliveira, Germison Silva Lopes, Ricardo Parente Garcia Vieira, Manoel Odorico de Moraes Filho, Maria Elisabete Amaral de Moraes, André Salim Khayat, Fabiano Cordeiro Moreira, Caroline Aquino Moreira-Nunes. Validation of Endogenous Control Genes by Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction for Acute Leukemia Gene Expression Studies. Genes. 2024 Jan 24;15(2)