Dataset Information

Evaluation of Reference Genes for Normalization of RT-qPCR Gene Expression Data for Trichoplusia ni Cells During Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus (AnpeNPV) Infection.

ABSTRACT: Baculovirus infection impacts global gene expression in the host cell, including the expression of housekeeping genes. Evaluation of candidate reference genes during a viral infection will inform the selection of appropriate reference gene(s) for the normalization of expression data generated by Reverse Transcription Quantitative Real-timePolymerase Chain Reaction (RT-qPCR). Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV) is able to infect the High Five cells (Tn-Hi5). In the present study, 10 candidate reference genes were evaluated in AnpeNPV-infected Tn-Hi5 cells. Gene expression data were analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. The candidate genes were further validated as reliable reference genes for RT-qPCR by analyzing the expression of three target genes. The results of data analysis using four statistical methods showed that RPS18 was the least stable among the candidate reference genes tested. 18S rRNA and 28S rRNA were not suitable as reference genes for RT-qPCR analysis in AnpeNPV-infected Tn-Hi5 cells. Comprehensive ranking of the 10 candidate reference genes by RefFinder analysis indicated that Ann B, c45128_g1, and ACT were the top three genes. Normalization of the expression of three target genes using the candidate reference genes indicated the same expression pattern when Ann B and c45128_g1 were used as reference genes, with slight differences in the relative expression at each infection time point. Overall, Ann B and c45128_g1 were recommended to be more suitable than the most commonly used reference genes, such as ACT, GAPDH, and TUB, for RT-qPCR data normalization in AnpeNPV-infected Tn-Hi5 cells up to 48 hpi.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC6324657 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Evaluation of Reference Genes for Normalization of RT-qPCR Gene Expression Data for Trichoplusia ni Cells During Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus (AnpeNPV) Infection.

Zhao Zhenjun Z Wang Linmei L Yue Dongmei D Ye Bo B Li Peipei P Zhang Bo B Fan Qi Q

Journal of insect science (Online) 20190101 1

Baculovirus infection impacts global gene expression in the host cell, including the expression of housekeeping genes. Evaluation of candidate reference genes during a viral infection will inform the selection of appropriate reference gene(s) for the normalization of expression data generated by Reverse Transcription Quantitative Real-timePolymerase Chain Reaction (RT-qPCR). Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV) is able to infect the High Five cells (Tn-Hi5). In the present ...[more]

PMID: 30624703

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Project description:The pupal diapause of univoltine Antheraea pernyi hampers sericultural and biotechnological applications, which requires a high eclosion incidence after artificial diapause termination to ensure production of enough eggs. The effect of pupal diapause termination using 20-hydroxyecdysone (20E) on the eclosion incidence has not been well-documented in A. pernyi. Here, the dosage of injected 20E was optimized to efficiently terminate pupal diapause of A. pernyi, showing that inappropriate dosage of 20E can cause pupal lethality and a low eclosion incidence. The optimal ratio of 20E to 1-month-old pupae was determined as 6 μg/g. Morphological changes showed visible tissue dissociation at 3 days post-injection (dpi) and eye pigmentation at 5 dpi. Comprehensive transcriptome analysis identified 1,355/1,592, 494/203, 584/297, and 1,238/1,404 upregulated and downregulated genes at 1, 3, 6, and 9 dpi, respectively. The 117 genes enriched in the information processing pathways of "signal transduction" and "signaling molecules and interaction" were upregulated at 1 and 3 dpi, including the genes involved in FOXO signaling pathway. One chitinase, three trehalase, and five cathepsin genes related to energy metabolism and tissue dissociation showed high expression levels at the early stage, which were different from the upregulated expression of four other chitinase genes at the later stage. Simultaneously, the expression of several genes involved in molting hormone biosynthesis was also activated between 1 and 3 dpi. qRT-PCR further verified the expression patterns of two ecdysone receptor genes (EcRB1 and USP) and four downstream response genes (E93, Br-C, βFTZ-F1, and cathepsin L) at the pupal and pharate stages, respectively. Taken together, these genes serve as a resource for unraveling the mechanism underlying pupal-adult transition; these findings facilitate rearing of larvae more than once a year and biotechnological development through efficient termination of pupal diapause in A. pernyi in approximately half a month.

Dataset Information

Evaluation of Reference Genes for Normalization of RT-qPCR Gene Expression Data for Trichoplusia ni Cells During Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus (AnpeNPV) Infection.

Publications

Evaluation of Reference Genes for Normalization of RT-qPCR Gene Expression Data for Trichoplusia ni Cells During Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus (AnpeNPV) Infection.

Similar Datasets

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