2.2. RT-qPCR Analysis

Insect pools were homogenized and total RNA was extracted employing TRIzol^® Reagent protocol (Life Technologies, Carlsbad, CA, USA) [18]. After being quantified by NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA), RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA) and an aliquot (1 μg) of each was used for first-strand cDNA synthesis with oligo dT (Promega), SuperScript^® II Reverse Transcriptase (Life Technologies), and RNaseOUT™ Recombinant Ribonuclease Inhibitor (Life Technologies). Power SYBR^® Green PCR Master Mix (Life Technologies) was used in quantitative PCR reactions carried out on an Applied Biosystems 7900HT Fast Real-Time PCR System, with the following cycle conditions: denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 15 s, annealing at 50–63 °C for 1 min, and extension at 60 °C for 1 min.

Forward and reverse primers were designed on target gene sequences deposited in GenBank (National Center for Biotechnology Information, NCBI) under the accession numbers provided in Table 1. Before being employed in qPCR analyses, each primer pair was tested by standard curve and dissociation curve analysis [19]. Tubulin and Glucose-6-phosphate dehydrogenase (G6PDH) were used as reference genes.

Primer pair sequences used to amplify Ceratitis capitata immune-related genes.

^a GenBank accession number of sequences used for primer design.