TY - JOUR
T1 - Evaluation of reference genes for gene expression studies using quantitative real-time PCR in Drosophila melanogaster after chemical exposures
AU - Kim, Yeong Ho
AU - Kim, Yi Seul
AU - Kim, Young Ho
N1 - Publisher Copyright:
© 2020
PY - 2020/6
Y1 - 2020/6
N2 - The fruit fly, Drosophila melanogaster, is mainly found in fermented and rotten fruits and is more tolerant to chemicals emitted during the fermentation process than other organisms. Its distinctive habitat suggests an evolutionary adaptation to the chemicals, such as acetic acid, ethanol, and 2-phenylethanol. Quantitative real-time PCR (qRT-PCR) assays can be performed to analyze the expression patterns of D. melanogaster genes putatively involved in chemical detoxification and metabolism, to better understand D. melanogaster adaptions to its environment. The selection of stably-expressed, internal reference genes across samples is crucial for accurate normalization of target gene expression parameters. In this study, we investigated the transcription levels of ten candidate reference genes: hsp22, ef1β, α-tub, rpL18, rpS3, argk, nd, tbp, gapdh, and ace, in D. melanogaster exposed to various concentrations of acetic acid, ethanol, and 2-phenylethanol, and expression stability of these genes was evaluated using three different programs: geNorm, NormFinder, and BestKeeper. These three software resulted in different stable genes, but suggested the selection of multiple reference genes for target gene normalization. For validation of reference genes, expression levels of adh were normalized with different references and combination of multiple genes. In the flies exposed to three chemicals, rpL18 was commonly suggested to be used for the most stable reference gene for comparing expression levels of genes potentially associated with environmental chemical tolerance in D. melanogaster.
AB - The fruit fly, Drosophila melanogaster, is mainly found in fermented and rotten fruits and is more tolerant to chemicals emitted during the fermentation process than other organisms. Its distinctive habitat suggests an evolutionary adaptation to the chemicals, such as acetic acid, ethanol, and 2-phenylethanol. Quantitative real-time PCR (qRT-PCR) assays can be performed to analyze the expression patterns of D. melanogaster genes putatively involved in chemical detoxification and metabolism, to better understand D. melanogaster adaptions to its environment. The selection of stably-expressed, internal reference genes across samples is crucial for accurate normalization of target gene expression parameters. In this study, we investigated the transcription levels of ten candidate reference genes: hsp22, ef1β, α-tub, rpL18, rpS3, argk, nd, tbp, gapdh, and ace, in D. melanogaster exposed to various concentrations of acetic acid, ethanol, and 2-phenylethanol, and expression stability of these genes was evaluated using three different programs: geNorm, NormFinder, and BestKeeper. These three software resulted in different stable genes, but suggested the selection of multiple reference genes for target gene normalization. For validation of reference genes, expression levels of adh were normalized with different references and combination of multiple genes. In the flies exposed to three chemicals, rpL18 was commonly suggested to be used for the most stable reference gene for comparing expression levels of genes potentially associated with environmental chemical tolerance in D. melanogaster.
KW - Acetic acid
KW - Drosophila melanogaster
KW - Ethanol, 2-phenylethanol
KW - Quantitative real-time PCR
KW - Reference gene
UR - http://www.scopus.com/inward/record.url?scp=85080896261&partnerID=8YFLogxK
U2 - 10.1016/j.aspen.2020.01.008
DO - 10.1016/j.aspen.2020.01.008
M3 - Article
AN - SCOPUS:85080896261
SN - 1226-8615
VL - 23
SP - 385
EP - 394
JO - Journal of Asia-Pacific Entomology
JF - Journal of Asia-Pacific Entomology
IS - 2
ER -