Suitable Reference Gene Selection for Different Strains and Developmental Stages of the Carmine Spider Mite,Tetranychus cinnabarinus, using Quantitative Real-Time PCR

Abstract Chilo partellus is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in C. partellus larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank ELF as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However, Tubulin and HSP70 were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of Chitinase and Acetylcholinesterase were normalized with the four most stable reference genes (ELF, HSP70, V-ATPase and Tubulin) and the least stable reference gene (18S and HSP70) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in C. partellus.

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Reference gene selection for quantitative real-time PCR normalization in different cherry genotypes, developmental stages and organs

Scientia Horticulturae ◽

10.1016/j.scienta.2014.10.027 ◽

2015 ◽

Vol 181 ◽

pp. 182-188 ◽

Cited By ~ 23

Author(s):

Xia Ye ◽

Fangming Zhang ◽

Yonghuan Tao ◽

Shangwei Song ◽

Jinbao Fang

Keyword(s):

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Gene Selection ◽

Developmental Stages ◽

Quantitative Real Time Pcr ◽

Reference Gene Selection ◽

Selection For

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Importance of Suitable Reference Gene Selection for Quantitative RT-PCR during ATDC5 Cells Chondrocyte Differentiation

PLoS ONE ◽

10.1371/journal.pone.0064786 ◽

2013 ◽

Vol 8 (5) ◽

pp. e64786 ◽

Cited By ~ 31

Author(s):

Zhichen Zhai ◽

Yongchang Yao ◽

Yingjun Wang

Keyword(s):

Reference Gene ◽

Gene Selection ◽

Suitable Reference Gene ◽

Chondrocyte Differentiation ◽

Rt Pcr ◽

Reference Gene Selection ◽

Selection For ◽

Suitable Reference

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Suitable reference gene selection for gene expression studies in knee osteoarthritis synovium using quantitative PCR analysis

Connective Tissue Research ◽

10.1080/03008207.2017.1391234 ◽

2017 ◽

Vol 59 (4) ◽

pp. 356-368 ◽

Cited By ~ 2

Author(s):

Hiroshi Watanabe ◽

Hirotaka Ishii ◽

Kenji Takahashi ◽

Shinro Takai ◽

Hitoshi Ozawa

Keyword(s):

Gene Expression ◽

Knee Osteoarthritis ◽

Quantitative Pcr ◽

Gene Selection ◽

Suitable Reference Gene ◽

Pcr Analysis ◽

Expression Studies ◽

Selection For ◽

Gene Expression Studies ◽

Suitable Reference

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Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Scientific Reports ◽

10.1038/s41598-021-92780-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Madhab Kumar Sen ◽

Kateřina Hamouzová ◽

Pavlina Košnarová ◽

Amit Roy ◽

Josef Soukup

Keyword(s):

Reference Gene ◽

Herbicide Resistance ◽

Reference Genes ◽

Gene Selection ◽

High Yield ◽

Suitable Reference Gene ◽

Grass Species ◽

Experimental Conditions ◽

Qrt Pcr ◽

Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

International Journal of Molecular Sciences ◽

10.3390/ijms19082258 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2258 ◽

Cited By ~ 16

Author(s):

Yuning Hu ◽

Hongtuo Fu ◽

Hui Qiao ◽

Shengming Sun ◽

Wenyi Zhang ◽

...

Keyword(s):

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Translation Initiation Factor ◽

Suitable Reference Gene ◽

Quantitative Real Time Pcr ◽

Macrobrachium Nipponense ◽

The Stability ◽

Suitable Reference

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

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