2.3.2 RNA Extraction, cDNA Synthesis and quantitative real-time
PCR (qRT-PCR)
The total RNA of each sample was extracted using the Invitrogen TRIzol
reagent (Invitrogen, Carlsbad, CA, USA) and RNA quantity and quality
were determined by a NanoDrop 2000 spectrophotometer (NanoDrop,
Wilmington, DE, USA). First-strand cDNA was synthesized with a FastQuant
RT Kit (with gDNase) (Tiangen Biotech, Beijing, China). The cDNA was
serially diluted (3-fold), and the dilutions were used to analyze the
qRT-PCR efficiency of the primers (Table S2). The β-actin andEF1-α genes of G. molesta were used as the internal
controls (Chen et al., 2014; Cao et al., 2015). The qRT-PCR mixture was
prepared as instructed by the SYBR ® Premix Ex TaqTM
(TliRNase H Plus) manual (Takara, Dalian, China). The reactions were
performed on a StepOne thermocycler (ABI, Carlsbad, USA) using the
two-step method and were analyzed with a melting curve analysis program.
The specificity of qRT-PCR primers was confirmed by the melting curve
and sequencing of the qRT-PCR products. The data were analyzed by using
the 2 -ΔΔCT method (Livak et al., 2001).