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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

Validation of the recommended reference gene.The expression profiles of the TSWV-receptor gene in nonviruliferous and viruliferous Frankliniella occidentalis were investigated using different combinations of reference genes. NF1, NF (1–2), NF (1–3), and NF9 indicate that expression of the TSWV-receptor gene was normalized using the best, top two, top three, and worst reference genes, respectively. The bar represents the mean and standard error of four biological replicates.
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pone.0135207.g003: Validation of the recommended reference gene.The expression profiles of the TSWV-receptor gene in nonviruliferous and viruliferous Frankliniella occidentalis were investigated using different combinations of reference genes. NF1, NF (1–2), NF (1–3), and NF9 indicate that expression of the TSWV-receptor gene was normalized using the best, top two, top three, and worst reference genes, respectively. The bar represents the mean and standard error of four biological replicates.

Mentions: Using one, two, or three of the best reference gene combinations for normalization, expression of the TSWV-receptor gene did not differ between viruliferous and nonviruliferous F. occidentalis (P > 0.05) (Fig 3).


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)

Validation of the recommended reference gene.The expression profiles of the TSWV-receptor gene in nonviruliferous and viruliferous Frankliniella occidentalis were investigated using different combinations of reference genes. NF1, NF (1–2), NF (1–3), and NF9 indicate that expression of the TSWV-receptor gene was normalized using the best, top two, top three, and worst reference genes, respectively. The bar represents the mean and standard error of four biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135207.g003: Validation of the recommended reference gene.The expression profiles of the TSWV-receptor gene in nonviruliferous and viruliferous Frankliniella occidentalis were investigated using different combinations of reference genes. NF1, NF (1–2), NF (1–3), and NF9 indicate that expression of the TSWV-receptor gene was normalized using the best, top two, top three, and worst reference genes, respectively. The bar represents the mean and standard error of four biological replicates.
Mentions: Using one, two, or three of the best reference gene combinations for normalization, expression of the TSWV-receptor gene did not differ between viruliferous and nonviruliferous F. occidentalis (P > 0.05) (Fig 3).

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