Quantification of Transgene-Derived Double-Stranded RNA in Plants Using the QuantiGene Nucleic Acid Detection Platform Toni A. Armstrong, † Hao Chen, ‡ Todd E. Ziegler, † Kelly R. Iyadurai, † Ai-Guo Gao, † Yongcheng Wang, † Zihong Song, ‡ Qing Tian, ‡ Qiang Zhang, † Jason M. Ward, ‡ Gerrit C. Segers, † Gregory R. Heck, ‡ and Jeffrey M. Staub* ,† † Monsanto Company, 700 Chesterfield Parkway North, St. Louis, Missouri 63017, United States ‡ Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, United States * S Supporting Information ABSTRACT: The expanding use of RNA interference (RNAi) in agricultural biotechnology necessitates tools for characterizing and quantifying double-stranded RNA (dsRNA)-containing transcripts that are expressed in transgenic plants. We sought to detect and quantify such transcripts in transgenic maize lines engineered to control western corn rootworm (Diabrotica virgifera virgifera LeConte) via overexpression of an inverted repeat sequence bearing a portion of the putative corn rootworm orthologue of yeast Snf7 (DvSnf7), an essential component of insect cell receptor sorting. A quantitative assay was developed to detect DvSnf7 sense strand-containing dsRNA transcripts that is based on the QuantiGene Plex 2.0 RNA assay platform from Affymetrix. The QuantiGene assay utilizes cooperative binding of multiple oligonucleotide probes with specificity for the target sequence resulting in exceptionally high assay specificity. Successful implementation of this assay required heat denaturation in the presence of the oligonucleotide probes prior to hybridization, presumably to dissociate primary transcripts carrying the duplex dsRNA structure. The dsRNA assay was validated using a strategy analogous to the rigorous enzyme-linked immunosorbent assay evaluations that are typically performed for foreign proteins expressed in transgenic plants. Validation studies indicated that the assay is sensitive (to 10 pg of dsRNA/g of fresh tissue), highly reproducible, and linear over ∼2.5 logs. The assay was validated using purified RNA from multiple maize tissue types, and studies indicate that the assay is also quantitative in crude tissue lysates. To the best of our knowledge, this is the first report of a non-polymerase chain reaction-based quantitative assay for dsRNA-containing transcripts, based on the use of the QuantiGene technology platform, and will broadly facilitate characterization of dsRNA in biological and environmental samples. KEYWORDS: dsRNA, DvSnf7, QuantiGene, RNAi, western corn rootworm ■ INTRODUCTION Gene silencing triggered by sequence-specific double-stranded RNA (dsRNA) has been applied by biotechnologists to control a wide range of target organisms. In transgenic plants, heterologous expression of primary transcripts that carry inverted repeat sequences capable of forming dsRNA molecules that target mRNAs encoding proteins with essential functions has been shown to have efficacy against a variety of pests, including nematodes, 1 lepidopteran larvae, 2 and coleopteran larvae, 3 in addition to fungal 4 and even parasitic plant targets. 5,6 Most recently, using diet bioassays, 3,7 it has been determined that when expressed in transgenic plants, a number of dsRNAs targeting mRNAs encoding proteins with essential functions in larvae of western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) provide effective control of this pest species. A number of factors can influence the efficacy of dsRNA- induced mortality in these insects, including dsRNA concen- tration and sequence length. 7,8 Attempts to dissect these parameters through diet bioassays have indicated that the LC 50 values (the concentration of dsRNA causing 50% mortality of the insect population) can depend on the amount of dsRNA fed to the insect. 7 To facilitate accurate quantification of transgene-derived dsRNA-containing transcripts, the develop- ment of new assay types capable of specific detection in complex plant or environmental samples is necessary. Approaches to the detection of dsRNA in biological samples include reverse transcription polymerase chain reaction (RT- PCR) or Northern blots. While denaturing Northern blots can resolve dsRNA species, this method is relatively low throughput, and the amounts of RNA can be challenging to quantify. On the other hand, RT-PCR can be high-throughput and can amplify through the secondary structure of inverted repeat-containing dsRNA transcripts if appropriately designed primers with a high annealing temperature are used. However, potential pitfalls of RT-PCR amplification of dsRNA have been described, including potential self-priming of RNA when it is expressed in both orientations that can interfere with template specificity 9 and the need for modifications to the standard RT- PCR method when strand-specific detection of dsRNA is desired. 10,11 Furthermore, for all RT-PCR applications, RNA purification is required to eliminate hybridization to cognate sequences in genomic DNA. Received: August 1, 2013 Revised: November 26, 2013 Accepted: December 2, 2013 Published: December 2, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 12557 dx.doi.org/10.1021/jf4031458 | J. Agric. Food Chem. 2013, 61, 12557-12564