Laboratory of Nematology, Wageningen Universiteit, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
BMC Genomics. 2011 Oct 17;12:510. doi: 10.1186/1471-2164-12-510.
RNAi technology by feeding of E. coli containing dsRNA in C. elegans has significantly contributed to further our understanding of many different fields, including genetics, molecular biology, developmental biology and functional genomics. Most of this research has been carried out in a single genotype or genetic background. However, RNAi effects in one genotype do not reveal the allelic effects that segregate in natural populations and contribute to phenotypic variation.
Here we present a method that allows for rapidly comparing RNAi effects among diverse genotypes at an improved high throughput rate. It is based on assessing the fitness of a population of worms by measuring the rate at which E. coli is consumed. Critically, we demonstrate the analytical power of this method by QTL mapping the loss of RNAi sensitivity (in the germline) in a recombinant inbred population derived from a cross between Bristol and a natural isolate from Hawaii. Hawaii has lost RNAi sensitivity in the germline. We found that polymorphisms in ppw-1 contribute to this loss of RNAi sensitivity, but that other loci are also likely to be important.
In summary, we have established a fast method that improves the throughput of RNAi in liquid, that generates quantitative data, that is easy to implement in most laboratories, and importantly that enables QTL mapping using RNAi.
通过在秀丽隐杆线虫中喂食含有 dsRNA 的大肠杆菌进行 RNAi 技术，极大地促进了我们对许多不同领域的理解，包括遗传学、分子生物学、发育生物学和功能基因组学。大多数研究都是在单一基因型或遗传背景下进行的。然而，一种基因型中的 RNAi 效应并不能揭示自然种群中分离的等位基因效应，也不能解释表型变异。
在这里，我们提出了一种方法，允许以提高的高通量率在不同基因型之间快速比较 RNAi 效应。它基于通过测量大肠杆菌消耗的速度来评估群体的适应性。至关重要的是，我们通过对来自布里斯托尔和来自夏威夷的自然分离株杂交产生的重组近交系群体中的生殖系 RNAi 敏感性丧失进行 QTL 作图，证明了这种方法的分析能力。夏威夷在生殖系中已经失去了 RNAi 敏感性。我们发现，ppw-1 中的多态性导致了这种 RNAi 敏感性的丧失，但其他基因座也可能很重要。
总之，我们建立了一种快速方法，提高了液体 RNAi 的通量，生成了定量数据，易于在大多数实验室中实施，并且重要的是，它能够使用 RNAi 进行 QTL 作图。
