Tissue specificity of immune-related gene expression in N. lugens. Total RNA was individually extracted from the salivary gland, fat body, gut and the remaining carcass of 5th instar nymphs. First-strand cDNA (20 ng) was analyzed in each qRT-PCR reaction. The reactions were performed with specific primers used to amplify (A) PGRP/GRP genes; (B) Toll genes; (C) CLIP genes; and (D) immune effector genes. The relative expression levels of each gene in each tissue were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values which were obtained from reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set at one. Three technical replications (n=3) were conducted and the ΔΔCt method was used to measure the relative transcript levels in tissues.