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Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis.

Yang C, Li H, Pan H, Ma Y, Zhang D, Liu Y, Zhang Z, Zheng C, Chu D - PLoS ONE (2015)

Bottom Line: To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial.Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis.This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.

View Article: PubMed Central - PubMed

Affiliation: Hunan Academy of Agricultural Sciences, Institute of Plant Protection, Changsha, Hunan, China.

ABSTRACT
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔCt method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.

No MeSH data available.


Related in: MedlinePlus

Determination of the optimal number of reference genes under plant virus stress.To determine the minimum number of genes required for normalization, the V-value was computed using geNorm. Starting with two genes, the software sequentially adds another gene and recalculates the NF ratio. If the added gene does not increase the NF ratio above the proposed 0.15 cut-off value, then the starting pair of genes is sufficient for normalization. If the NF ratio is adequately increased, more genes should be incorporated.
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pone.0135207.g002: Determination of the optimal number of reference genes under plant virus stress.To determine the minimum number of genes required for normalization, the V-value was computed using geNorm. Starting with two genes, the software sequentially adds another gene and recalculates the NF ratio. If the added gene does not increase the NF ratio above the proposed 0.15 cut-off value, then the starting pair of genes is sufficient for normalization. If the NF ratio is adequately increased, more genes should be incorporated.

Mentions: Under plant virus stress, the first V-value less than 0.15 was after V2/3 (Fig 2). This means that two reference genes were sufficient for reliable normalization regardless of the virus infection status of the insect.


Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis.

Yang C, Li H, Pan H, Ma Y, Zhang D, Liu Y, Zhang Z, Zheng C, Chu D - PLoS ONE (2015)

Determination of the optimal number of reference genes under plant virus stress.To determine the minimum number of genes required for normalization, the V-value was computed using geNorm. Starting with two genes, the software sequentially adds another gene and recalculates the NF ratio. If the added gene does not increase the NF ratio above the proposed 0.15 cut-off value, then the starting pair of genes is sufficient for normalization. If the NF ratio is adequately increased, more genes should be incorporated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526564&req=5

pone.0135207.g002: Determination of the optimal number of reference genes under plant virus stress.To determine the minimum number of genes required for normalization, the V-value was computed using geNorm. Starting with two genes, the software sequentially adds another gene and recalculates the NF ratio. If the added gene does not increase the NF ratio above the proposed 0.15 cut-off value, then the starting pair of genes is sufficient for normalization. If the NF ratio is adequately increased, more genes should be incorporated.
Mentions: Under plant virus stress, the first V-value less than 0.15 was after V2/3 (Fig 2). This means that two reference genes were sufficient for reliable normalization regardless of the virus infection status of the insect.

Bottom Line: To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial.Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis.This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.

View Article: PubMed Central - PubMed

Affiliation: Hunan Academy of Agricultural Sciences, Institute of Plant Protection, Changsha, Hunan, China.

ABSTRACT
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔCt method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.

No MeSH data available.


Related in: MedlinePlus