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Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid, Lipaphis erysimi (Kalt).

Koramutla MK, Aminedi R, Bhattacharya R - Sci Rep (2016)

Bottom Line: Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests.Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi.This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.

View Article: PubMed Central - PubMed

Affiliation: ICAR-National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute Campus, New Delhi-110012, India.

ABSTRACT
Mustard aphid, also known as turnip aphid (Lipaphis erysimi) is a major insect pest of rapeseed-mustard group of crops. Tremendous economic significance has led to substantial basic research involving gene-expression studies in this insect species. In qRT-PCR analysis of gene-expression, normalization of data against RNA variation by using appropriate reference gene is fundamental. However, appropriate reference genes are not known in case of L. erysimi. We evaluated 11 candidate reference genes for their expression stability in 21 samples of L. erysimi subjected to various regimes of experimental treatments. Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests. In the validated samples, expression stability of the reference genes was analysed by employing four different statistical softwares geNorm, NormFinder, BestKeeper and deltaCt. Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi. This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.

No MeSH data available.


Related in: MedlinePlus

Whisker box plots of reference gene expression in 5 treatments.The boxes represent the mean Ct values and the line in the box represents median. The bars across the boxes show the minimum and maximum Ct values. Whiskers represent 95% confidence intervals. Data represent mean values ± SD (3n = 9).
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f3: Whisker box plots of reference gene expression in 5 treatments.The boxes represent the mean Ct values and the line in the box represents median. The bars across the boxes show the minimum and maximum Ct values. Whiskers represent 95% confidence intervals. Data represent mean values ± SD (3n = 9).

Mentions: In qRT-PCR, a variable Ct value of all the reference genes across the 5 treatments indicated that their gene-expression levels are affected by the treatments albeit at differential extents (Fig. 3). The expression of SDHB, RPL29, and AK were maximally affected by the developmental stages and temperature stress. Similarly, the expression of EF1A, GAPDH, AK, and SDHB varied significantly among the samples collected from aphids either fed on artificial diet for different durations or treated with sinigrin in the diet. In a comparative assessment, a narrow range of variable Ct values in case of 16S, RPS18 and RPL13 across the type and time course of the treatments empirically suggested their relatively more stability of expression. In contrary, RPL29, EF1A and GAPDH displayed highest variations in Ct values when all the experimental treatments were taken into account. Irrespective of the treatments, 16S rRNA was found to be most abundant, with the lowest mean Ct value 9.23, compared to the transcript levels of other protein coding genes (Fig. 3).


Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid, Lipaphis erysimi (Kalt).

Koramutla MK, Aminedi R, Bhattacharya R - Sci Rep (2016)

Whisker box plots of reference gene expression in 5 treatments.The boxes represent the mean Ct values and the line in the box represents median. The bars across the boxes show the minimum and maximum Ct values. Whiskers represent 95% confidence intervals. Data represent mean values ± SD (3n = 9).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4863174&req=5

f3: Whisker box plots of reference gene expression in 5 treatments.The boxes represent the mean Ct values and the line in the box represents median. The bars across the boxes show the minimum and maximum Ct values. Whiskers represent 95% confidence intervals. Data represent mean values ± SD (3n = 9).
Mentions: In qRT-PCR, a variable Ct value of all the reference genes across the 5 treatments indicated that their gene-expression levels are affected by the treatments albeit at differential extents (Fig. 3). The expression of SDHB, RPL29, and AK were maximally affected by the developmental stages and temperature stress. Similarly, the expression of EF1A, GAPDH, AK, and SDHB varied significantly among the samples collected from aphids either fed on artificial diet for different durations or treated with sinigrin in the diet. In a comparative assessment, a narrow range of variable Ct values in case of 16S, RPS18 and RPL13 across the type and time course of the treatments empirically suggested their relatively more stability of expression. In contrary, RPL29, EF1A and GAPDH displayed highest variations in Ct values when all the experimental treatments were taken into account. Irrespective of the treatments, 16S rRNA was found to be most abundant, with the lowest mean Ct value 9.23, compared to the transcript levels of other protein coding genes (Fig. 3).

Bottom Line: Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests.Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi.This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.

View Article: PubMed Central - PubMed

Affiliation: ICAR-National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute Campus, New Delhi-110012, India.

ABSTRACT
Mustard aphid, also known as turnip aphid (Lipaphis erysimi) is a major insect pest of rapeseed-mustard group of crops. Tremendous economic significance has led to substantial basic research involving gene-expression studies in this insect species. In qRT-PCR analysis of gene-expression, normalization of data against RNA variation by using appropriate reference gene is fundamental. However, appropriate reference genes are not known in case of L. erysimi. We evaluated 11 candidate reference genes for their expression stability in 21 samples of L. erysimi subjected to various regimes of experimental treatments. Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests. In the validated samples, expression stability of the reference genes was analysed by employing four different statistical softwares geNorm, NormFinder, BestKeeper and deltaCt. Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi. This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.

No MeSH data available.


Related in: MedlinePlus