배경: PHB는 세포 내 신호전달을 조절하고, 핵내에서 전사 활성화와 세포주기를 조절한다. 따라서 PHB 이상발현은 종양발생, 진행 및 만성 대사성질환이나 염증성질환과 관련이 있음이 알려지고 있다. 본 연구에서는 PHB1 및 PHB2 유전자 정량발현 one-step RT-qPCR키트를 개발하여 이의 검사성능을 평가하여 보았다.
방법: TaqMan법을 적용하여 one-step RT-qPCR키트를 개발하였다. 정상인 20명의 말초혈액검체, 급성골수성백혈병 20예, 만성골수성백혈병 13예, 급성림프구성백혈병 7예의 진단시 골수검체와 상품화된 인간유래 RNA 관리물질을 이용하여 개발된 키트의 검사능 평가에 이용하였다.
결과: 개발한 PHB1 및 PHB2 RT-qPCR 키트의 측정내 정밀도와 측정 간 정밀도는 모두 2% 이내의 매우 양호한 정밀도를 보였다. 급성골수성백혈병, 만성골수성백혈병 및 급성림프구성백혈병환자에서 PHB1 유전자 발현량은 각각 0.898-0.993, 0.817-0.976, 0.844-1.074이었고, PHB2 유전자 발현량의 분포는 각각 0.957-1.024, 0.988-1.047, 0.937-1.059이었다. 개발한 PHB1 및 PHB2 유전자 정량 발현량 검사키트의 백혈병에 대한 민감도, 특이도, 양성예측률, 음성예측률 및 검사효능은 각각에서 50% 이상이었다.
결론: 본 연구에서 개발한 PHB1 및 PHB2 유전자 정량발현 one-step RT-qPCR 검사키트는 백혈병 등 혈액암의 진단 및 잔류병소 측정뿐만 아니라, 비만 등 만성대사/염증성질환이나 당뇨병 등의 추적검사 및 새로운 병태생리 규명을 위한 연구에 유용하게 사용될수 있을 것으로 판단된다.
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Original Article
Prohibitin 유전자 발현양 검출 역전사 중합효소연쇄반응 키트 개발 및 검사성능 평가
Development and Performance Evaluation of a Quantitative Reverse Transcription PCR Kit for the Determination of prohibitin Gene Expression
1전남대학교 의과대학 두뇌한국21 플러스사업
2광양사랑병원 진단검사의학과
3전남대학교 공과대학 전자컴퓨터공학부
4동신대학교 한의과대학
5화순전남대학교병원 진단검사의학과·전남대학교 의과대학 진단검사의학교실
Bo-Ram Na, M.S.1, Young-Eun Lee, M.S.1, Min-Gu Kang, M.D.2, Yonggwan Won, Ph.D.3, Hye-Ran Kim, Ph.D.4, Myung-Geun Shin, M.D. 
1,5
1,51Brain Korea 21 Plus Project, Chonnam National University Medical School, Gwangju, Korea
2Department of Laboratory Medicine, GwangYang Sarang General Hospital, GwangYang, Korea
3School of Electronics and Computer Engineering, College of Engineering, Chonnam National University, Gwangju, Korea
4College of Korean Medicine, Dongshin University, Naju, Korea
5Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hwasun Hospital, Hwasun, Korea
Received: July 31, 2019; Revised: October 29, 2019; Accepted: October 29, 2019
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lab Med Online 2020; 10(3): 214-220
Published July 1, 2020 https://doi.org/10.3343/lmo.2020.10.3.214
Copyright © The Korean Society for Laboratory Medicine.
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.
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.
Keywords
Prohibitin, Gene expression, Quantitative reverse transcription PCR (RT-qPCR), Laboratory performance
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