Selection of reference genes for tissue/organ samples on day 3 fifth‐instar larvae in silkworm, <i>Bombyx mori</i>

Wang, Genhong; Chen, Yanfei; Zhang, Xiaoying; Bai, Bingchuan; Yan, Hao; Qin, Daoyuan; Xia, Qingyou

doi:10.1002/arch.21458

Cited by 7 publications

(7 citation statements)

References 56 publications

(65 reference statements)

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“…AK, a type of phosphagen kinase that is widely distributed in various tissues of invertebrates, exhibits high stability between different tissues of Drosophila suzukii and Bombus lucorum [50,51]. Eukaryotic translation initiation factor (EIF) is involved in the initiation of eukaryotic translation and is the most stable gene among different tissues of Bombyx mori [52]. UBC and EF1-α are also commonly used as reference genes [22,35], but in our study, these genes were not as stable as were ribosomal protein genes.…”

Section: Discussionmentioning

confidence: 99%

Selection of reference genes for tissue/organ samples of adults of Eucryptorrhynchus scrobiculatus

2020

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Eucryptorrhynchus scrobiculatus is an important wood-boring pest of Ailanthus altissima in China, where it causes a large number of these trees to weaken or even die. To identify genes related to economic traits or specific cellular processes in E. scrobiculatus, gene expression in multiple tissue/organ samples is commonly surveyed, and reference genes are required in this process as a control for normalization. In the present study, 18 candidate reference genes from E. scrobiculatus were identified, and the expression levels of these reference genes were estimated through quantitative real-time PCR. Differences in expression levels were analyzed with four algorithms (geNorm, NormFinder, BestKeeper and delta Ct) and comprehensively with RefFinder. With the most stable levels of expression in different tissues, RPL13, RPS3 and RPL36 were determined to be suitable for use as candidate reference genes. Moreover, the expression profile of one target gene (glycoside hydrolase family 45, GH45) confirmed the reliability of the selected candidate reference genes. This study provides the first set of suitable candidate reference genes for gene expression studies in E. scrobiculatus, and the findings will facilitate subsequent transcriptomics and functional gene research on this pest.

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Section: Discussionmentioning

confidence: 99%

Selection of reference genes for tissue/organ samples of adults of Eucryptorrhynchus scrobiculatus

2020

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“…The total RNA was treated with RNase-Free DNase I (#M6101, Promega, USA) and then reverse transcribed into cDNA using M-MLV Reverse Transcriptase (#M1701, Promega, USA). The cDNA temples of different developmental stages were used for RT-PCR with primers BmPGRP2-1qRT, BmPGRP2-2qRT, and internal control TIF-4A (53, 57–59), of which the amplification cycles was 30, 30, and 25, respectively. The cDNA of each tissue was used for qPCR analysis with primers BmPGRP2-1qRT and BmPGRP2-2qRT on an ABI Prism 7500 (Applied Biosystems, USA) using an SYBR Premix Ex Taq II (#RR820A, TaKaRa, China).…”

Section: Methodsmentioning

confidence: 99%

“…The cDNA of each tissue was used for qPCR analysis with primers BmPGRP2-1qRT and BmPGRP2-2qRT on an ABI Prism 7500 (Applied Biosystems, USA) using an SYBR Premix Ex Taq II (#RR820A, TaKaRa, China). The thermal program of qPCR consisted of 95°C for 30 s, 40 cycles at 95°C for 5 s and 60°C for 30 s, 95°C for 15 s, 60°C for 60 s, and melt for 15 s. The control TIF-4A was used for qPCR analysis of gene expression level to standardize the variance among the different templates (53, 57–59). Each detection was performed thrice.…”

Section: Methodsmentioning

confidence: 99%

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Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses

et al. 2019

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Peptidoglycan recognition protein (PGRP) is an important pattern recognition receptor in innate immunity that is vital for bacterial recognition and defense in insects. Few studies report the role of PGRP in viral infection. Here we cloned two forms of PGRP from the model lepidopteran Bombyx mori : BmPGRP2-1 is a transmembrane protein, whereas BmPGRP2-2 is an intracellular protein. BmPGRP2-1 bound to diaminopimelic acid (DAP)-type peptidoglycan (PGN) to activate the canonical immune deficiency (Imd) pathway. BmPGRP2-2 knockdown reduced B. mori nucleopolyhedrovirus (BmNPV) multiplication and mortality in cell lines and in silkworm larvae, while its overexpression increased viral replication. Transcriptome and quantitative PCR (qPCR) results confirmed that BmPGRP2 negatively regulated phosphatase and tensin homolog ( PTEN ). BmPGRP2-2 expression was induced by BmNPV, and the protein suppressed PTEN-phosphoinositide 3-kinase (PI3K)/Akt signaling to inhibit cell apoptosis, suggesting that BmNPV modulates BmPGRP2-2-PTEN-PI3K/Akt signaling to evade host antiviral defense. These results demonstrate that the two forms of BmPGRP2 have different functions in host responses to bacteria and viruses.

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“…Besides RNA yield, quality, and reverse transcription efficiency [ 4 , 7 , 14 ], it is also necessary to apply a data normalization strategy to eliminate the variations caused by technology or experiment. At present, the geNorm, NormFinder, and BestKeeper software tools are primarily used to evaluate the expression stability of candidate RGs, and to identify the minimum number of RGs needed under diverse experimental conditions [ 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

Zhao¹,

Cheng²,

Zhang³

et al. 2021

Animals

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As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characterization of the gene expression profiles of genes of interest is essential to the exploration of the intrinsic regulatory mechanisms of rumen development in goats. Thus, the selection of suitable reference genes (RGs) is an important prerequisite for real-time quantitative PCR (RT-qPCR). In the present study, 16 candidate RGs were identified from our previous transcriptome sequencing of caprine rumen tissues. The quantitative expressions of the candidate RGs were measured using the RT-qPCR method, and the expression stability of the RGs was assessed using the geNorm, NormFinder, and BestKeeper programs. GeNorm analysis showed that the M values were less than 0.5 for all the RGs except GAPT4, indicating that they were stably expressed in the rumen tissues throughout development. RPS4X and RPS6 were the two most stable RGs. Furthermore, the expressions of two randomly selected target genes (IGF1 and TOP2A), normalized by the selected most stable RGs (RPS4X and RPS6), were consistent with the results of RNA sequencing, while the use of GAPDH and ACTB as RGs resulted in altered profiles. Overall, RPS4X and RPS6 showed the highest expression stability and the lowest coefficients of variation, and could be used as the optimal reference combination for quantifying gene expression in rumen tissues via RT-qPCR analysis.

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Selection of reference genes for tissue/organ samples on day 3 fifth‐instar larvae in silkworm, Bombyx mori

Cited by 7 publications

References 56 publications

Selection of reference genes for tissue/organ samples of adults of Eucryptorrhynchus scrobiculatus

Selection of reference genes for tissue/organ samples of adults of Eucryptorrhynchus scrobiculatus

Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses

Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

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