Development and Performance Evaluation of a Quantitative Reverse Transcription PCR Kit for the Determination of prohibitin Gene Expression

ABSTRACT

Background@#Prohibitin (PHB) regulates intracellular signal pathways, transcription, and cell cycles. Aberrant expression of the PHB gene is known to be related totumorigenesis, tumor progression, and chronic metabolic and inflammatory diseases. The present study aimed to develop a one-step quantitative reverse transcription PCR (RT-qPCR) kit for quantifying PHB mRNA levels and evaluate its performance in the laboratory. @*Methods@#TaqMan chemistry was used to develop the one-step PHB1 and PHB2 RT-qPCR kit. Normal peripheral blood cells from healthy individuals (N=20) and leukemia cells from patients initially diagnosed with acute myeloid leukemia (AML, N=20), chronic myeloid leukemia (CML, N=13), and acute lymphoid leukemia (ALL, N=7) were enrolled to evaluate the laboratory performance of the kit using commercially available total human RNA controls. @*Results@#The intra-assay and inter-assay precision of the kit developed in this study was less than 2%. The distribution of PHB1 mRNA expression of AML, CML, and ALL was 0.898-0.993 (median 0.936), 0.817-0.976 (0.918), and 0.844-1.074 (0.973), respectively. The distribution of PHB2 mRNA expression of AML, CML, and ALL was 0.957-1.024 (median 0.985), 0.988-1.047 (1.002), and 0.937-1.059 (1.004), respectively. The sensitivity, specificity, positive and negative predictive value, and test effectiveness of the developed PHB1 and PHB2 kit were greater than 50% for each parameter. @*Conclusions@#Our developed kit would be useful for diagnosing leukemia as well as detecting residual disease. Additionally, this kit could be used for monitoring and conducting molecular pathophysiological studies of obesity, metabolic, and inflammatory diseases.