Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants
The development of genetically-engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2016-11-01
|
| Series: | Frontiers in Plant Science |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fpls.2016.01672/full |
| _version_ | 1811243351627792384 |
|---|---|
| author | Chunxiao Yang Chunxiao Yang Chunxiao Yang Evan L. Preisser Hongjun Zhang Hongjun Zhang Yong Liu Liangying Dai Huipeng Pan Huipeng Pan Xuguo Zhou |
| author_facet | Chunxiao Yang Chunxiao Yang Chunxiao Yang Evan L. Preisser Hongjun Zhang Hongjun Zhang Yong Liu Liangying Dai Huipeng Pan Huipeng Pan Xuguo Zhou |
| author_sort | Chunxiao Yang |
| collection | DOAJ |
| description | The development of genetically-engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is RT-qPCR, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly-used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔCt method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended respectively for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants. |
| first_indexed | 2024-04-12T14:05:54Z |
| format | Article |
| id | doaj.art-c103a17aec774be2ad5bd6f09007e6ec |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-12T14:05:54Z |
| publishDate | 2016-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-c103a17aec774be2ad5bd6f09007e6ec2022-12-22T03:30:05ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2016-11-01710.3389/fpls.2016.01672226445Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plantsChunxiao Yang0Chunxiao Yang1Chunxiao Yang2Evan L. Preisser3Hongjun Zhang4Hongjun Zhang5Yong Liu6Liangying Dai7Huipeng Pan8Huipeng Pan9Xuguo Zhou10College of Plant Protection, Hunan Agricultural UniversityInstitute of Plant Protection, Hunan Academy of Agricultural SciencesUniversity of KentuckyDepartment of Biological Sciences, University of Rhode IslandUniversity of KentuckyInstitute for the Control of Agrochemicals, Ministry of AgricultureInstitute of Plant Protection, Hunan Academy of Agricultural SciencesCollege of Plant Protection, Hunan Agricultural UniversityUniversity of KentuckySouth China Agricultural UniversityUniversity of KentuckyThe development of genetically-engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is RT-qPCR, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly-used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔCt method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended respectively for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.http://journal.frontiersin.org/Journal/10.3389/fpls.2016.01672/fullEnvironmental Risk Assessmentreference geneCoccinella septempunctataRT-qPCR analysisPlant incorporated protectantRNAi-transgenic plants |
| spellingShingle | Chunxiao Yang Chunxiao Yang Chunxiao Yang Evan L. Preisser Hongjun Zhang Hongjun Zhang Yong Liu Liangying Dai Huipeng Pan Huipeng Pan Xuguo Zhou Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants Frontiers in Plant Science Environmental Risk Assessment reference gene Coccinella septempunctata RT-qPCR analysis Plant incorporated protectant RNAi-transgenic plants |
| title | Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants |
| title_full | Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants |
| title_fullStr | Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants |
| title_full_unstemmed | Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants |
| title_short | Selection of reference genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of RNAi transgenic plants |
| title_sort | selection of reference genes for rt qpcr analysis in coccinella septempunctata to assess un intended effects of rnai transgenic plants |
| topic | Environmental Risk Assessment reference gene Coccinella septempunctata RT-qPCR analysis Plant incorporated protectant RNAi-transgenic plants |
| url | http://journal.frontiersin.org/Journal/10.3389/fpls.2016.01672/full |
| work_keys_str_mv | AT chunxiaoyang selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT chunxiaoyang selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT chunxiaoyang selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT evanlpreisser selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT hongjunzhang selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT hongjunzhang selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT yongliu selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT liangyingdai selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT huipengpan selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT huipengpan selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants AT xuguozhou selectionofreferencegenesforrtqpcranalysisincoccinellaseptempunctatatoassessunintendedeffectsofrnaitransgenicplants |