Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115.
G3 (Bethesda). 2013 Sep 4;3(9):1607-16. doi: 10.1534/g3.113.007021.
The evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo.
评估特定的内源性转录本水平对于理解转录调控非常重要。更具体地说，它有助于独立确认使用微阵列分析或 RNA-seq 获得的结果，并评估 RNA 干扰 (RNAi) 介导的基因敲低。设计用于高质量、中等通量转录本水平评估（即定量、实时 PCR (qPCR)）的特异性和有效的引物并非易事。为了满足社区的需求，已经设计并提供了用于哺乳动物基因的预定义 qPCR 引物对，例如通过 PrimerBank。在这项工作中，我们对用于哺乳动物 PrimerBank 的算法进行了改编和完善，设计了用于 13860 个果蝇基因的 45417 对引物，每个基因有 3 对或更多引物对。我们使用 SYBR Green 基 qPCR 和来自常规 PCR 的产物的序列分析，对从早期果蝇胚胎中表达的约 300 个随机选择的基因的引物对进行了实验验证。所有相关信息，包括引物序列、异构体特异性、空间转录靶向以及任何可用的验证结果和/或用户反馈，都可从在线数据库 (www.flyrnai.org/flyprimerbank) 获得。在 FlyPrimerBank 中，研究人员可以一次检索一个基因的引物信息，也可以批量检索。重要的是，我们包括了每个预测扩增序列与来自几个公共资源的 RNAi 试剂的重叠，这使得研究人员可以选择适合 RNAi 试剂敲低评估的引物（即避免扩增 RNAi 试剂本身）。我们展示了该资源在体内验证 RNAi 试剂方面的实用性。
