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

Author:

Pessoa Flávia Melo Cunha de Pinho1,Viana Vitória Beatriz de Jesus2,de Oliveira Marcelo Braga2,Nogueira Beatriz Maria Dias1,Ribeiro Rodrigo Monteiro3,Oliveira Deivide de Sousa13,Lopes Germison Silva4ORCID,Vieira Ricardo Parente Garcia5,de Moraes Filho Manoel Odorico1,de Moraes Maria Elisabete Amaral1,Khayat André Salim2ORCID,Moreira Fabiano Cordeiro2ORCID,Moreira-Nunes Caroline Aquino126ORCID

Affiliation:

1. Department of Medicine, Pharmacogenetics Laboratory, Drug Research and Development Center (NPDM), Federal University of Ceará, Fortaleza 60430-275, CE, Brazil

2. Department of Biological Sciences, Oncology Research Center, Federal University of Pará, Belém 66073-005, PA, Brazil

3. Department of Hematology, Fortaleza General Hospital (HGF), Fortaleza 60150-160, CE, Brazil

4. Department of Hematology, César Cals General Hospital, Fortaleza 60015-152, CE, Brazil

5. Department of Hematology, São Vicente de Paulo Maternity Hospital, Barbalha 63180-000, CE, Brazil

6. Central Unity, Molecular Biology Laboratory, Clementino Fraga Group, Fortaleza 60115-170, CE, Brazil

Abstract

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.

Funder

National Council of Technological and Scientific Development

PROPESP/UFPA

Publisher

MDPI AG

Subject

Genetics (clinical),Genetics

Reference115 articles.

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4. Seifi, M., Ghasemi, A., Heidarzadeh, S., Khosravi, M., Namipashaki, A., Soofiany, V.M., Khosroshahi, A.A., and Danaei, N. (2012). Polymerase Chain Reaction, IntechOpen.

5. Loftis, A.D., and Reeves, W.K. (2012). Veterinary PCR Diagnosis, Bentham Science Publishers.

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