Lab Med Online.  2020 Jul;10(3):214-220. 10.3343/lmo.2020.10.3.214.

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

• Na BR
• Lee YE
• Kang MG
• Won Y
• Kim HR
• Shin MG

• KMID: 2504131
• DOI: http://doi.org/10.3343/lmo.2020.10.3.214

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.

Keyword

Prohibitin; Gene expression; Quantitative reverse transcription PCR (RT-qPCR); Laboratory performance

Figure

• 

  Fig. 1 Amplification plots of standards for ABL (upper panel), PHB1 (middle panel), and PHB2 (lower panel) genes. Five standards of ABL, PHB1, and PHB2 genes were used in each experiment with the same number of copies (Standard 1: 2×102 copies/μL, Standard 2: 2×103 copies/μL, Standard 3: 2×104 copies/μL, Standard 4: 2×105 copies/μL, Standard 5: 2×106 copies/μL).

• 

  Fig. 2 Theslope of the standard curve used toestimate PCR amplification efficiency of ABL (upper), PHB1 (middle), and PHB2 (lower) genes. A standard curve is graphically represented as a semi-log regression line plot of the Cq value versus log of standard nucleic acid copies.

• 

  Fig. 3 Distribution of PHB1 and PHB2 mRNAexpressionin healthy individuals and individuals with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoid leukemia (ALL).*, P<0.05.

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