Selection and Evaluation of Reference Genes for Expression Analysis Using qRT-PCR inChilo suppressalis(Lepidoptera: Pyralidae)

Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) is a valuable tool for estimating gene expression; however, the validity is largely dependent on the selection of stable reference genes. The suitability of various reference genes for qRT-PCR analysis was evaluated in, Chilo suppressalis (Walker). The ΔCt method, geNorm, NormFinder, and BestKeeper were used to evaluate the suitability of nine candidate reference genes for normalizing gene expression in larval tissues and organs and during high and low temperature stress. The ΔCt method, geNorm, and NormFinder produced similar stability rankings; H3, UBI, and EF1 were the most stable reference genes for monitoring gene expression in larval tissue and organs, and EF1, TUB, and AK were the optimal genes for thermal stress. However, for thermal stress, RPS11 was the most stable gene based on BestKeeper. To validate these recommendations, the expression profile of the gene encoding heat shock protein 60 (Hsp60) was investigated. Hsp60 transcript levels showed significant differences when normalized to the most versus least stable reference genes. These results further confirm the importance of testing reference genes using the selected experimental parameters. The reference genes identified in the present study will improve the quality of gene expression data obtained for C. suppressalis and will facilitate future studies aimed at understanding the biology of this important insect pest.

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“…Fluorescent quantitative (q)PCR was performed using an ABI7500 instrument (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the instructions of the Sybr Premix Ex Taq II Kit (Takara, Kyoto, Japan), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal reference. The 2 2DDCt method was applied to calculate the relative mRNA expression of the target gene: DDC t = DC t experimental group -DC t control group and DC t = C t target gene -C t internal reference (28).…”

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“…The reaction conditions were as follows: predenaturation at 95 °C for 30 s; 45 cycles of denaturation at 95 °C for 30 s, annealing for 20 s and extension at 72 °C for 30 s. Next, the expression of ZEB1-AS1, miR-129-5p, ZEB1, Bcl-2 and MDR1 was determined. The ratio of the target gene expression between the experimental group and the control group was expressed based on the 2 −ΔΔCt method and the formula was: ΔΔCT = ΔCt experimental group − ΔCt control group [27]. Ct denoted the number of amplification cycles when the real time fluorescence intensity reached the set threshold, and the amplification was denoted in the logarithmic growth phase.…”

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