Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli)

Ma, Liman; Wang, Wenji; Liu, Conghui; Yu, Hao; Wang, Zhigang; Wang, Xubo; Qi, Jie; Zhang, Quanqi

doi:10.1016/j.margen.2013.08.002

Cited by 72 publications

(24 citation statements)

References 33 publications

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“…The high expression level of 18S rRNA in R. padi was expected, in agreement with the lowest Cq value in other insects [44]. EF-1α, a well-known HKG in various organisms [51] and an evolutionarily conserved nuclear gene in aphids [52], [53], [54], can be used to classify the population of R. padi into various subgroups depending on EF-1α diversity in the population [52], [53]. Its expression level was consistent between winged aphids and wingless aphids, lower than that of 18S rRNA but slightly higher than that of ACT1 and GAPDH.…”

Section: Resultssupporting

confidence: 73%

Sequencing and Validation of Reference Genes to Analyze Endogenous Gene Expression and Quantify Yellow Dwarf Viruses Using RT-qPCR in Viruliferous Rhopalosiphum padi

Liu

Mar

et al. 2014

PLoS ONE

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The bird cherry-oat aphid (Rhopalosiphum padi), an important pest of cereal crops, not only directly sucks sap from plants, but also transmits a number of plant viruses, collectively the yellow dwarf viruses (YDVs). For quantifying changes in gene expression in vector aphids, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a touchstone method, but the selection and validation of housekeeping genes (HKGs) as reference genes to normalize the expression level of endogenous genes of the vector and for exogenous genes of the virus in the aphids is critical to obtaining valid results. Such an assessment has not been done, however, for R. padi and YDVs. Here, we tested three algorithms (GeNorm, NormFinder and BestKeeper) to assess the suitability of candidate reference genes (EF-1α, ACT1, GAPDH, 18S rRNA) in 6 combinations of YDV and vector aphid morph. EF-1α and ACT1 together or in combination with GAPDH or with GAPDH and 18S rRNA could confidently be used to normalize virus titre and expression levels of endogenous genes in winged or wingless R. padi infected with Barley yellow dwarf virus isolates (BYDV)-PAV and BYDV-GAV. The use of only one reference gene, whether the most stably expressed (EF-1α) or the least stably expressed (18S rRNA), was not adequate for obtaining valid relative expression data from the RT-qPCR. Because of discrepancies among values for changes in relative expression obtained using 3 regions of the same gene, different regions of an endogenous aphid gene, including each terminus and the middle, should be analyzed at the same time with RT-qPCR. Our results highlight the necessity of choosing the best reference genes to obtain valid experimental data and provide several HKGs for relative quantification of virus titre in YDV-viruliferous aphids.

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Section: Resultssupporting

confidence: 73%

Sequencing and Validation of Reference Genes to Analyze Endogenous Gene Expression and Quantify Yellow Dwarf Viruses Using RT-qPCR in Viruliferous Rhopalosiphum padi

Liu

Mar

et al. 2014

PLoS ONE

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“…Although ribosomal genes were rarely used as internal controls, recent studies began to realize that they were suitable for many cases. For example, RPL17 was identified as an appropriate reference gene in the black rockfish (Sebastes schlegeli) among different tissues at larval developmental stages (Ma et al, 2013). Similarly, the expression of RPL17 did not vary in the European sea bass (Dicentrarchus labrax) collected from marine and freshwater habitats (Varsamos et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

“…However, considering the complexity, economic cost and work load, the practical solution is the use of top three stable genes when greater than or equal to three reference genes were recommended by geNorm, and such a strategy has been widely employed in many studies (e.g. Ma et al, 2013;Sun et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

Reference gene selection for quantitative gene expression studies during biological invasions: A test on multiple genes and tissues in a model ascidian Ciona savignyi

Huang

Gao

Jiang

et al. 2016

Gene

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“…Primer pairs for these genes were carefully designed to ensure amplification of specific PCR products from reverse transcribed cDNA. The presence of a single amplicon peak in the melting curve analysis (Figure S3) further confirmed the amplification of a specific PCR product [20], [47], [48]. Across all the available datasets (total samples, MA2 and MA8 tissue culture lines, different media treatments), PD00380 and PD00569 were ranked as the top two most stably expressed genes by geNorm (Figure 2, Figure S2).…”

Section: Discussionmentioning

confidence: 53%

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

et al. 2014

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BackgroundThe somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels.ResultsIn this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization.ConclusionsSystematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

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Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli)

Cited by 72 publications

References 33 publications

Sequencing and Validation of Reference Genes to Analyze Endogenous Gene Expression and Quantify Yellow Dwarf Viruses Using RT-qPCR in Viruliferous Rhopalosiphum padi

Sequencing and Validation of Reference Genes to Analyze Endogenous Gene Expression and Quantify Yellow Dwarf Viruses Using RT-qPCR in Viruliferous Rhopalosiphum padi

Reference gene selection for quantitative gene expression studies during biological invasions: A test on multiple genes and tissues in a model ascidian Ciona savignyi

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

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