Responsive expressions to bacterial infection of immune-related genes in N. lugens nymphs. Fifth instar nymphs were microinjected with E. coli K12 or B. subtilis. Total RNA was extracted from the nymphs at the indicated times after injection. PBS-injected samples were used as controls. First-strand cDNA (20 ng) was analyzed in each real-time quantitative PCR reaction. The reactions were performed with specific primers for amplifying PGRP/GRP genes, immune effector genes and Toll genes. The relative expression levels of each gene at different time points were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values, which were obtained for reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set to one. Three technical replications (n=3) were conducted and the relative transcript levels at each time point were calculated using the ΔΔCt method. The E. coli K12- and B. subtilis injected samples are shown on the left (black) and right (dark gray), respectively. C refers to the PBS-injected control. 6, 12, and 24 h refer to RNA extracted from bacteria-injected nymphs at 6, 12, and 24 h p.i.