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RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops

Overview of attention for article published in BMC Biotechnology, January 2018
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Title
RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops
Published in
BMC Biotechnology, January 2018
DOI 10.1186/s12896-018-0413-6
Pubmed ID
Authors

Po-Hao Wang, Greg Schulenberg, Shannon Whitlock, Andrew Worden, Ning Zhou, Stephen Novak, Wei Chen

Abstract

RNA interference (RNAi) technology has been widely used to knockdown target genes via post-transcriptional silencing. In plants, RNAi is used as an effective tool with diverse applications being developed such as resistance against insects, fungi, viruses, and metabolism manipulation. To develop genetically modified (GM) RNAi traits for insect control, a transgene is created and composed of an inversely-repeated sequence of the target gene with a spacer region inserted between the repeats. The transgene design is subject to form a self-complementary hairpin RNA (hpRNA) and the active molecules are > 60 bp doubled-stranded RNA (dsRNA) derived from the hpRNA. However, in some cases, an undesirable intermediate such as single-stranded RNA (ssRNA) may be formed, which is not an active molecule. The aforementioned characteristics of RNAi traits lead to increase the challenges for RNAi-derived dsRNA quantitation. To quantify the dsRNA and distinguish it from the ssRNA in transgenic maize, an analytical tool is required to be able to effectively quantify dsRNA which contains a strong secondary structure. Herein, we develop a modified qRT-PCR method (abbreviated as RNase If -qPCR) coupled with a ssRNA preferred endonuclease (i.e., RNase If). This method enables the precise measurement of the active molecules (i.e., dsRNA) derived from RNAi traits of GM crops and separately quantifies the dsRNA from ssRNA. Notably, we also demonstrate that the RNase If -qPCR is comparable to a hybridization-based method (Quantigene Plex 2.0). To our best knowledge, this is the first report of a method combining RNase If with modified qRT-PCR protocol. The method represents a reliable analytical tool to quantify dsRNA for GM RNAi crops. It provides a cost-effective and feasible analytical tool for general molecular laboratory without using additional equipment for other methods. The RNase If -qPCR method demonstrates high sensitivity (to 0.001 pg/ μL of dsRNA), precision and accuracy. In this report, we demonstrated the deployment of this method to characterize the RNAi events carrying v-ATPase C in maize during trait development process. The method can be utilized in any application which requires the dsRNA quantification such as double-stranded RNA virus or sprayable dsRNA as herbicide.

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Mendeley readers

The data shown below were compiled from readership statistics for 24 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 24 100%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 6 25%
Researcher 6 25%
Student > Master 3 13%
Other 2 8%
Student > Doctoral Student 1 4%
Other 1 4%
Unknown 5 21%
Readers by discipline Count As %
Agricultural and Biological Sciences 11 46%
Biochemistry, Genetics and Molecular Biology 4 17%
Earth and Planetary Sciences 1 4%
Arts and Humanities 1 4%
Neuroscience 1 4%
Other 2 8%
Unknown 4 17%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 23 January 2018.
All research outputs
#9,931,944
of 12,406,609 outputs
Outputs from BMC Biotechnology
#556
of 697 outputs
Outputs of similar age
#245,890
of 339,559 outputs
Outputs of similar age from BMC Biotechnology
#1
of 1 outputs
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