Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica
Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica
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DOI：
10.1016/j.jinsphys.2012.05.013
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发表时间：
2013-02-01
影响因子：
2.2
通讯作者：
Reynolds, Stuart E.
中科院分区：
文献类型：
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作者：
Garbutt, Jennie S.;Belles, Xavier;Reynolds, Stuart E.
RNA干扰（RNA interference，RNAi）是一种由双链RNA（double-stranded RNA，dsRNA）介导的特异性基因沉默机制，已成为昆虫反向遗传学研究的重要工具。然而，不幸的是，这种技术受到昆虫物种对RNAi的可变敏感性的限制。我们提出，昆虫血淋巴中的dsRNA的快速降解可能会阻碍RNAi的基因沉默，并通过实验研究了两种昆虫中dsRNA持续存在的动力学，烟草天蛾Manduca sexta，这是一种实验困难的物种，已经经历了RNAi方案和德国蟑螂，德国小蠊，这是已知的对实验RNAi高度敏感。离体测定显示，dsRNA被M中的酶快速降解。sexta血淋巴血浆中，而dsRNA在B中持续时间更长。德国等离子体。基于定量逆转录PCR的分析显示，dsRNA相应地从M. sexta体内血淋巴。分枝sexta dsRNAse在高温下失活，并被EDTA抑制。这些发现使我们提出，dsRNA在昆虫血淋巴中的持续存在率（由一种或多种核酸酶的作用介导）可能是决定昆虫物种对RNAi易感性的重要因素。(C)2012爱思唯尔有限公司保留所有权利。
RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi. (C) 2012 Elsevier Ltd. All rights reserved.