Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.
The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.
Laboratory of Pesticide, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Miao
M
Yuan
Laboratory of Pesticide, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Yanhui
Y
Lu
Laboratory of Pesticide, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Xun
X
Zhu
Laboratory of Pesticide, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Hu
H
Wan
Laboratory of Pesticide, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Muhammad
M
Shakeel
Laboratory of Pesticide, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Sha
S
Zhan
Laboratory of Insect Molecular Biology and Biotechnology, Department of Applied Biology, College of Natural Resources and Life Science, Dong-A University, Busan, Korea.
Byung-Rae
BR
Jin
Laboratory of Pesticide, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Jianhong
J
Li
D000818:Animals; D004032:Diet; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D017344:Genes, Insect / Q000235:genetics*; D006430:Hemiptera / Q000187:drug effects / Q000235:genetics*; D012275:Oryza / Q000187:drug effects / Q000235:genetics; D010575:Pesticides / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012639:Seeds / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D013312:Stress, Physiological; D013696:Temperature
D010575:Pesticides; D012333:RNA, Messenger