Evaluation of Quantitative Real-Time Pcr Reference Genes for the Investigation of Gene Expression Profiles in Honeybee Developmental Stages

Carposina sasakii is one of the most important pests on the quality of stone and pome fruits. Investigation of a gene expression level in the species is hampered because of the gap of validated reference genes. The expression variation in the transcription levels of eight candidate reference genes, Actin (ACT), Tubulinbeta-1 (TUB), Ribosomal protein 49 (RP49), Elongation factor1-alpha (EF-1a), Elongation factor1-b (EF-1b), Elongation factor1-d (EF-1d), Ribosomal proteinL13 (RPL13) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were analyzed by quantitative real-time PCR (qPCR). The stability and ranking of these gene expression profiles in three organ types (head, thorax and abdomen), three developmental stages (larva, pupa and moth), and five diapause states (non-diapause, pre-diapause, diapause 0 d, diapause 20 d and diapause 60 d) were assessed using two algorithm-based methods, geNorm and NormFinder. EF-1a, ACT and GAPDH were evaluated to be the three stable reference genes based on the important observations and comprehensive analysis, whereas TUB and EF-1b showed low expression stability. Best gene combinations for different qPCR analysis in C. sasakii could be chosen from the three stable reference genes, the using of two reference genes is sufficient to effectively normalize qPCR data in C. sasakii. The study laid the foundation for gene expression analysis in C. sasakii and provided new information for the selection of reference genes.

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Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

Genome ◽

10.1139/gen-2017-0176 ◽

2018 ◽

Vol 61 (5) ◽

pp. 349-358 ◽

Cited By ~ 6

Author(s):

Yanchun You ◽

Miao Xie ◽

Liette Vasseur ◽

Minsheng You

Keyword(s):

Gene Expression ◽

Real Time ◽

Plutella Xylostella ◽

Real Time Pcr ◽

Reference Genes ◽

Developmental Stages ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Expression Studies ◽

Gene Expression Studies

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

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Validation of quantitative real-time PCR reference genes for the determination of seasonal and labor-specific gene expression profiles in the head of Western honey bee, Apis mellifera

PLoS ONE ◽

10.1371/journal.pone.0200369 ◽

2018 ◽

Vol 13 (7) ◽

pp. e0200369 ◽

Cited By ~ 12

Author(s):

KyungHwan Moon ◽

Si Hyeock Lee ◽

Young Ho Kim

Keyword(s):

Gene Expression ◽

Apis Mellifera ◽

Honey Bee ◽

Real Time Pcr ◽

Reference Genes ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Specific Gene ◽

Quantitative Real Time Pcr

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Evaluation and Validation of Reference Genes for Quantitative Real-Time PCR in Helopeltis theivora Waterhouse (Hemiptera: Miridae)

Scientific Reports ◽

10.1038/s41598-019-49479-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Zheng Wang ◽

Qianqian Meng ◽

Xi Zhu ◽

Shiwei Sun ◽

Shengfeng Gao ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Developmental Stages ◽

Transcriptional Level ◽

Quantitative Real Time Pcr ◽

Systematic Analysis ◽

Helopeltis Theivora ◽

Qrt Pcr

Abstract Helopeltis theivora Waterhouse is a predominant sucking pest in many tropic economic crops, such as tea, cocoa and coffee. Quantitative real-time PCR (qRT-PCR) is one of the most powerful tools to analyze the gene expression level and investigate the mechanism of insect physiology at transcriptional level. Gene expression studies utilizing qRT-PCR have been applied to numerous insects so far. However, no universal reference genes could be used for H. theivora. To obtain accurate and reliable normalized data in H. theivora, twelve candidate reference genes were examined under different tissues, developmental stages and sexes by using geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder algorithms, respectively. The results revealed that the ideal reference genes differed across the treatments, and the consensus rankings generated from stability values provided by these programs suggested a combination of two genes for normalization. To be specific, RPS3A and Actin were the best suitable reference genes for tissues, RPL13A and GAPDH were suitable for developmental stages, EF1α and RPL13A were suitable for sexes, and RPL13A and RPS3A were suitable for all samples. This study represents the first systematic analysis of reference genes for qRT-PCR experiments in H. theivora, and the results can provide a credible normalization for qRT-PCR data, facilitating transcript profiling studies of functional genes in this insect.

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Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Mung Bean under Abiotic Stress and Cercospora canescens Infection

Legume Research - An International Journal ◽

10.18805/lr-507 ◽

2020 ◽

Author(s):

X. W. Ke ◽

L. H. Yin ◽

J. Xu ◽

W. N. Sun ◽

X. D. Xu ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Mung Bean ◽

Expression Profiles ◽

Pcr Analysis ◽

Saline Stress ◽

Stable Gene ◽

Quantitative Real Time Pcr

The objective of the study was to identify suitable reference genes that can be used for quantitative real-time PCR (qPCR) analysis in mung bean (Vigna radiata). Therefore, 10 potential reference genes were selected and the results showed that ubiquitin-conjugating enzyme was suitable as reference under drought and pathogen infection stress; elongation factor 1-á was the most stable gene under waterlogging; and actin performed the best under saline stress. These selected reference genes were further confirmed by analysis of the expression profiles of catalase and peroxidase under waterlogging. Our results will contribute to the improvement of the accuracy of gene expression evaluation in mung bean.

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