Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

ABSTRACT

BACKGROUND: The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene. METHODS AND RESULTS: The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases. CONCLUSIONS: It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.