Development of broadly targeted human endogenous gammaretroviral pol-based real time PCRs quantitation of RNA expression in human tissues
Development of broadly targeted human endogenous gammaretroviral pol-based real time PCRs quantitation of RNA expression in human tissues
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DOI：
10.1016/j.jviromet.2005.04.016
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发表时间：
2005-10-01
影响因子：
3.1
通讯作者：
Blomberg, J
中科院分区：
文献类型：
--
作者：
Forsman, A;Yun, ZH;Blomberg, J
内源性逆转录病毒序列（ERVs）是一种动态的基因组成分，对基因表达和基因组结构有着深远的影响。其表达的程度并不为人所知。构建了几种广泛靶向的实时逆转录PCR（QPCR）系统，用于监测人γ逆转录病毒ERV主要组群的RNA表达。pol基因的高度保守的逆转录酶（RT）和整合酶（IN）结构域被用作PCR的靶标，PCR是基于探针的（TaqMan（R））和无探针的（SYBR（R）绿色）。测试了不同水平的引物和探针简并性，有或没有肌苷。几个PCR具有每个PCR反应几个HERV核酸拷贝的灵敏度。特异性大致与引物和探针拟合的预期一致。在不同的人体组织中研究了γ逆转录病毒HERV RNA的表达。每个HERV组具有特定的表达模式。HERV-E在睾丸中高表达，HERV-I/T在脑和睾丸中高表达，HERV-H在脑和睾丸中高表达，HERV-W在胎盘中高表达。在50名献血者的血浆和20人的唾液中未检测到γ逆转录病毒RNA。总之，创建了一套用于研究γ逆转录病毒HERV RNA表达的工具。(c)2005 Elsevier B. V.保留所有权利。
Endogenous retroviral sequences (ERVs) are dynamic genomic components with profound influences on gene expression and genomic structure. Their extent of expression is not well known. Several broadly targeted real-time reverse transcription PCR (QPCRs) systems for surveillance of RNA expression of the major groups of human gammaretroviral ERVs were constructed. The highly conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were used as targets for the PCRs, which were both probe-based (TaqMan (R)) and probe-less (SYBR (R) Green). Different levels of primer and probe degeneracy, with or without inosine, were tested. Several of the PCRs had sensitivities of a few HERV nucleic acid copies per PCR reaction. Specificities were approximately as expected from the fit of primers and probes. Gammaretroviral HERV RNA expression was studied in different human tissues. Each HERV group had a specific pattern of expression. HERV-E was highly expressed in testis, HERV-I/T in brain and testis, HERV-H in brain and testis, while HERV-W was highly expressed in placenta. Gammaretroviral RNA was not detected in plasma from 50 blood donors in saliva from 20 persons. In conclusion, a set of tools for investigation of gammaretroviral HERV RNA expression was created. (c) 2005 Elsevier B.V. All rights reserved.