RNAi knockdown of NMD in D Melanogaster

Two core factors of the NMD machinery in D. melanogaster, Upf1 and Upf2, were knocked down using RNAi, as described in Rehwinkel et al. (2005) RNA, PMID: 16199763. Each of the two knockdowns were compared to mock RNAi knockdowns as described in Blanchette et al. (2005) Genes Dev, PMID: 15937219 in a dual channel experiment, using a custom splice-junction microarray design, see Blanchette et al. (2005), PMID: 15937219. The aim of the experiment was to identify which isoforms of alternatively spliced genes were affected by NMD knockdown and thereby gain insight into the NMD mechanism in Drosophila. The samples were hybridized in a dual channel setup, to custom designed Splice-Junction arrays manufactured by Agilent. A total of 6 independent knockdowns (3 Upf1 and 3 Upf2) were generated and hybridized to 6 different arrays. On each array a control sample was hybridized as well. The control sample is a pool of 3 independent mock RNAi knockdowns.

本研究采用RNA干扰（RNA interference, RNAi）技术敲低黑腹果蝇（Drosophila melanogaster, D. melanogaster）无义介导的mRNA降解（Nonsense-mediated mRNA Decay, NMD）通路的两个核心因子Upf1与Upf2，具体实验方法参照Rehwinkel等人2005年发表于*RNA*期刊的研究（PMID: 16199763）。本实验采用双通道设计，将两组敲低样本分别与空白RNAi对照（mock RNAi）敲低样本进行比较，实验方案参照Blanchette等人2005年发表于*Genes & Development*的研究（PMID: 15937219），所用芯片为定制化剪接接头微阵列（splice-junction microarray），相关设计细节亦可参阅Blanchette等人2005年的上述研究（PMID: 15937219）。本实验的核心目标为鉴定受NMD敲低影响的可变剪接基因异构体（isoform），进而解析黑腹果蝇中的NMD作用机制。所有样本采用双通道方式进行杂交，所用芯片为安捷伦（Agilent）定制化剪接接头微阵列。本研究共构建6组独立的敲低样本（3组Upf1敲低与3组Upf2敲低），并将其分别与6张不同的芯片进行杂交；每张芯片同时设置对照样本，该对照样本为3组独立的空白RNAi对照敲低样本的混合池。