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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

Variation in AP-1, MYR and Hsp83 gene-expression data normalized by different reference genes and their combinations.Fold change in AP-1, MYR and Hsp83 transcripts in aphids subjected to treatments of developmental stage (A), sinigrin (B) and temperature stress (C), respectively. Stable reference genes 16S, RPS18, RPL13, their combinations and least stable reference gene RPL29 were independently used as normalizer. Values represent mean ± SE (3n = 9) and comparison of means was carried out by student’s t test (P < 0.05). Different letters indicate significantly different values.
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f6: Variation in AP-1, MYR and Hsp83 gene-expression data normalized by different reference genes and their combinations.Fold change in AP-1, MYR and Hsp83 transcripts in aphids subjected to treatments of developmental stage (A), sinigrin (B) and temperature stress (C), respectively. Stable reference genes 16S, RPS18, RPL13, their combinations and least stable reference gene RPL29 were independently used as normalizer. Values represent mean ± SE (3n = 9) and comparison of means was carried out by student’s t test (P < 0.05). Different letters indicate significantly different values.

Mentions: It was apparent that the best ranked reference gene for one treatment was not applicable to the other treatments. Therefore, for experimental convenience it was imperative to identify the reference genes that show acceptable, if not the highest, stability in expression within and across the treatments. Analysis of consolidated data indicated 16S, RPS18, and RPL13 as the top ranked reference genes in terms of stability in gene-expression for most of the treatment conditions. In contrary, RPL29 was found to be the least stable. Applicability of these reference genes in normalization, either as single or in combination, was tested. This was carried out by assessing the transcript level of AP-1, MYR and Hsp83 in the treatments of developmental stage, sinigrin and heat stress, respectively (Fig. 6). In case of developmental stage, either of RPS18 and RPL13 or combination of them as normalizer showed higher consistency in qRT-PCR data compared to normalization by 16S and its combinations (Fig. 6A). However, for sinigrin treatment and heat stress normalization by a single recommended gene produced consistent and comparable result (Fig. 6B,C). In contrary, RPL29 being the least stable reference gene when used as normalizer showed significant variation in the results (Fig. 6).


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)

Variation in AP-1, MYR and Hsp83 gene-expression data normalized by different reference genes and their combinations.Fold change in AP-1, MYR and Hsp83 transcripts in aphids subjected to treatments of developmental stage (A), sinigrin (B) and temperature stress (C), respectively. Stable reference genes 16S, RPS18, RPL13, their combinations and least stable reference gene RPL29 were independently used as normalizer. Values represent mean ± SE (3n = 9) and comparison of means was carried out by student’s t test (P < 0.05). Different letters indicate significantly different values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Variation in AP-1, MYR and Hsp83 gene-expression data normalized by different reference genes and their combinations.Fold change in AP-1, MYR and Hsp83 transcripts in aphids subjected to treatments of developmental stage (A), sinigrin (B) and temperature stress (C), respectively. Stable reference genes 16S, RPS18, RPL13, their combinations and least stable reference gene RPL29 were independently used as normalizer. Values represent mean ± SE (3n = 9) and comparison of means was carried out by student’s t test (P < 0.05). Different letters indicate significantly different values.
Mentions: It was apparent that the best ranked reference gene for one treatment was not applicable to the other treatments. Therefore, for experimental convenience it was imperative to identify the reference genes that show acceptable, if not the highest, stability in expression within and across the treatments. Analysis of consolidated data indicated 16S, RPS18, and RPL13 as the top ranked reference genes in terms of stability in gene-expression for most of the treatment conditions. In contrary, RPL29 was found to be the least stable. Applicability of these reference genes in normalization, either as single or in combination, was tested. This was carried out by assessing the transcript level of AP-1, MYR and Hsp83 in the treatments of developmental stage, sinigrin and heat stress, respectively (Fig. 6). In case of developmental stage, either of RPS18 and RPL13 or combination of them as normalizer showed higher consistency in qRT-PCR data compared to normalization by 16S and its combinations (Fig. 6A). However, for sinigrin treatment and heat stress normalization by a single recommended gene produced consistent and comparable result (Fig. 6B,C). In contrary, RPL29 being the least stable reference gene when used as normalizer showed significant variation in the results (Fig. 6).

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