昆虫小器官RNA荧光原位杂交技术

In situ fluorescent RNA hybridization in insect small organs

【目的】在昆虫基因表达和功能研究中,RNA原位杂交技术越来越受到青睐。该技术不仅能定性定量反应基因表达的时空特异性,而且能在细胞水平上检测基因表达的调控模式。为了将该技术更好地在昆虫小器官研究中运用,我们以果蝇幼虫翅芽为例优化了改技术。【方法】解剖果蝇3龄幼虫翅芽进行原位杂交实验。【结果】我们发现影响原位杂交结果的因素十分复杂,包括取材时期,探针的合成,预杂交/杂交的时间和温度,清洗时间,适当的对照等。通过RNA荧光原位杂交实验,我们揭示了调控细胞记忆的trithorax基因在3龄翅芽广泛表达,并且受到转录因子Optomotor-blind的负调控。【结论】这一技术方法为研究昆虫小器官的基因表达和调控提供了便捷手段。

[Objectives] RNA in situ hybridization is a popular technique for analyzing the expression and function of insect genes. This technique provides both qualitative and quantitative data on not only the spatio-temporal expression patterns of genes, but also on their cellular regulation. To optimize this technique, we performed RNA in situ fluorescent hybridization assays using the Drosophila larval wing disc as a representative insect small organ. [Methods] Wing discs of 3rd instar Drosophila larvae were dissected and used in RNA in situ hybridization. [Results[ Many factors can influence the results of in situ hybridization, including the developmental stage of insect organs, probes, time and temperature of pre-hybridization and hybridization, washing time, and proper controls. We found that the cell memory gene trithorax is ubiquitously expressed in the wing disc and negatively regulated by the transcription factor Optomotor-blind. [Conclusion] In situ RNA hybridization is a convenient tool for revealing the expression patterns and regulation of genes in small organs of insects.