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      Selection of reference genes for expression analysis of plant-derived microRNAs in Plutella xylostella using qRT-PCR and ddPCR

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          Abstract

          The establishment of an expression quantification system that can be easily applied for the comparison of microRNAs (miRNAs) from biological samples is an important step toward understanding functional mechanisms in organisms. However, there is lack of attention on the selection of reference genes for miRNA expression profiling in insect herbivores. Here, we explored the candidate reference genes in a notorious pest of cruciferous crops, Plutella xylostella, for normalization of miRNA expression in developmental stages and tissues and in response to a change of food source from artificial diet to host plant Arabidopsis thaliana. We first compared the expression levels and stability of eight small RNAs using qRT-PCR, and found that miR11 was the most suitable reference gene for expression quantification of the miRNAs. We then confirmed this finding using digital droplet PCR and further validated with a well-studied cross-kingdom miRNA derived from A. thaliana (ath-miR159a). However, none of the reference genes was applicable for all experimental conditions, and multiple reference genes were sometimes required within the same experiment. Our work provides a method for the selection of reference genes for quantification of plant-derived miRNAs, which paves the way for unveiling their roles in the insect-plant coevolution.

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          Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

          Background Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. Results Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). Conclusion Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.
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            MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments

            The conclusions of thousands of peer-reviewed publications rely on data obtained using fluorescence-based quantitative real-time PCR technology. However, the inadequate reporting of experimental detail, combined with the frequent use of flawed protocols is leading to the publication of papers that may not be technically appropriate. We take the view that this problem requires the delineation of a more transparent and comprehensive reporting policy from scientific journals. This editorial aims to provide practical guidance for the incorporation of absolute minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis) and guidance on the publication of pure 'reference gene' articles.
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              Diamondback moth ecology and management: problems, progress, and prospects.

              Agricultural intensification and greater production of Brassica vegetable and oilseed crops over the past two decades have increased the pest status of the diamondback moth (DBM), Plutella xylostella L., and it is now estimated to cost the world economy US$4-5 billion annually. Our understanding of some fundamental aspects of DBM biology and ecology, particularly host plant relationships, tritrophic interactions, and migration, has improved considerably but knowledge of other aspects, e.g., its global distribution and relative abundance, remains surprisingly limited. Biological control still focuses almost exclusively on a few species of hymenopteran parasitoids. Although these can be remarkably effective, insecticides continue to form the basis of management; their inappropriate use disrupts parasitoids and has resulted in field resistance to all available products. Improved ecological understanding and the availability of a series of highly effective selective insecticides throughout the 1990s provided the basis for sustainable and economically viable integrated pest management (IPM) approaches. However, repeated reversion to scheduled insecticide applications has resulted in resistance to these and more recently introduced compounds and the breakdown of IPM programs. Proven technologies for the sustainable management of DBM currently exist, but overcoming the barriers to their sustained adoption remains an enormous challenge.
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                Author and article information

                Contributors
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: VisualizationRole: Writing – original draft
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: Software
                Role: Methodology
                Role: Writing – review & editing
                Role: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                1 August 2019
                2019
                : 14
                : 8
                : e0220475
                Affiliations
                [1 ] State Key Laboratory for Ecological Pest Control of Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou, China
                [2 ] International Joint Research Laboratory of Ecological Pest Control, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, China
                [3 ] Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada
                Chinese Academy of Agricultural Sciences Institute of Plant Protection, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-8659-3123
                http://orcid.org/0000-0001-9042-6432
                Article
                PONE-D-19-08092
                10.1371/journal.pone.0220475
                6675394
                31369618
                2331237a-6467-4c6d-9818-b76e49480552
                © 2019 Zhang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 March 2019
                : 16 July 2019
                Page count
                Figures: 7, Tables: 2, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003392, Natural Science Foundation of Fujian Province;
                Award ID: 2019J01369
                Award Recipient :
                Funded by: National Key R&D Program of China
                Award ID: 2017YFD0200400
                Award Recipient :
                Funded by: Fujian Provincial Science and Technology Major Project
                Award ID: 2018NZ0002-1
                Award Recipient :
                This study was funded by the National Key R&D Program of China (grant number 2017YFD0200400 awarded to Assoc. Prof. Weiyi He), the Fujian Provincial Science and Technology Major Project (grant number 2018NZ0002-1 awarded to Prof. Minsheng You), and the Natural Science Foundation of Fujian Province (grant number 2019J01369 awarded to Assoc. Prof. Weiyi He). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Natural antisense transcripts
                MicroRNAs
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                MicroRNAs
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and Life Sciences
                Developmental Biology
                Life Cycles
                Larvae
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Biochemistry
                Lipids
                Fats
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Model Organisms
                Arabidopsis Thaliana
                Research and Analysis Methods
                Model Organisms
                Arabidopsis Thaliana
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Brassica
                Arabidopsis Thaliana
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Plant and Algal Models
                Arabidopsis Thaliana
                Biology and Life Sciences
                Biophysics
                Nucleic Acid Thermodynamics
                Genetic Annealing
                Physical Sciences
                Physics
                Biophysics
                Nucleic Acid Thermodynamics
                Genetic Annealing
                Biology and Life Sciences
                Biochemistry
                Nucleic Acids
                Nucleic Acid Thermodynamics
                Genetic Annealing
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Arthropoda
                Insects
                Custom metadata
                All relevant data are within the manuscript and its Supporting Information files.

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