Computational protocol: Silencing of the olfactory co-receptor gene in Dendroctonus armandi leads to EAG response declining to major host volatiles

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Protocol publication

[…] The transcripts of different tissues, developments, and RNAi treated adults were measured by using a CFX-96 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the Roche SYBR Green system (Roche Diagnostics GmbH, SandhoferStraße, Mannheim, Germany). Actin gene (GenBank accession number: KJ507199.1) of D. armandi was used as endogenous control to normalize the target gene expression. The primers of the target and reference genes were designed by Primer Express 5.0 (Applied Biosystems, Carlsbad, CA) (). qRT-PCR reactions were conducted in 20 μL reaction mixtures, each containing 10 μL of 2 × SYBR Premix Ex Taq (Roche Diagnostics GmbH, Sandhofer Straße, Mannheim, Germany), 0.3 μL of each primer (10 μM), 1 μL of cDNA, and 8.4 μL of sterilized H2O. The qRT-PCR cycling conditions were as follows: 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 20 s and 72 °C for 20 s; melt curves stages at 95 °C for 15 s; 60 °C for 1 min; and 95 °C for 15 s. Experiments for test samples, endogenous control, and negative control were performed in triplicate to ensure reproducibility. Relative quantification was performed by using the comparative 2–ΔΔCt method. All data were normalized to endogenous actin levels from the same tissue samples. [...] Data from qRT-PCR and EAG tests were analyzed using SPSS 17.0 (IBM SPSS Statistics, Chicago, IL, USA). ANOVA and Duncan’s new multiple range test (P = 0.05) were used to determine whether differences in DarmOrco mRNA levels or EAG responses were significant among different treatment groups. […]

Pipeline specifications

Software tools Primer Express, SPSS
Applications Miscellaneous, qPCR, Non-coding RNA analysis
Organisms Davidina armandi