Quantification of transgene-derived double-stranded RNA in plants using the QuantiGene nucleic acid detection platform.

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.

A novel real-time polymerase chain reaction method for high throughput quantification of small regulatory RNAs.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are important players of both transcriptional and post-transcriptional gene silencing networks. In order to investigate the functions of these small regulatory RNAs, a system with high sensitivity and specificity is desperately needed to quantitatively detect their expression levels in cells and tissues. However, their short length of 19-24 nucleotides and strong similarity between related species render most conventional expression analysis methods ineffective. Here we describe a novel primer for small RNA-specific reverse transcription and a new TaqMan technology-based real-time method for quantification of small RNAs. This method is capable of quantifying miRNA and siRNA in the femtomolar range, which is equivalent to ten copies per cell or fewer. The assay has a high dynamic range and provides linear readout of miRNA concentrations that span seven orders of magnitude and allows us to discriminate small RNAs that differ by as little as one nucleotide. Using the new method, we investigated the expression pattern of gma-miRMON1, a novel miRNA identified from soybean leaves. The results were consistent with our results obtained from Northern blot analysis of gma-miRMON1 and Affymetrix microarray analysis of the gma-miRMON1 precursor, suggesting that the new method can be used in transcription profiling.

Modifying lysine biosynthesis and catabolism in corn with a single bifunctional expression/silencing transgene cassette.

Although it is one of the major crops in the world, corn has poor nutritional quality for human and animal consumption due to its low lysine content. Here, we report a method of simultaneous expression of a deregulated lysine biosynthetic enzyme, CordapA, and reduction of a bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharophine dehydrogenase (LKR/SDH), in transgenic corn plants by a single transgene cassette. This is accomplished by inserting an inverted-repeat sequence targeting the maize LKR/SDH gene into an intron of a transgene cassette that expresses CordapA. This combination of LKR/SDH silencing and CordapA expression led to the accumulation of free lysine to over 4000 p.p.m. in transgenic corn grain, compared to less than 100 p.p.m. in wild-type controls. This intron-embedded silencing cassette design reduces the number of transgene cassettes needed in transgenic approaches for manipulating metabolic pathways that sometimes require expression of one gene and silencing of another.