Quantitative PCR Analysis of Insect Tissues

Total RNA from hemocyte, fat body or midgut was isolated following tissue collection by the protocol described above. RNA was extracted from each tissue in TRIzol (Life Technologies) using phase lock gel tubes (Quanta Bio), and isolated using manufacturer protocols. The concentration and quality of RNA samples was assessed using a NanoDrop spectrophotometer (Thermo Scientific) and cDNAs were synthesized from the corresponding RNA samples using the QuantiTech Reverse Transcription Kit (Qiagen). Synthesized cDNA samples were used as templates for quantitative PCR assays using DyNAmo HS SYBR green qPCR kits (Thermo Fisher Scientific) and gene-specific primers (S1 Table). Prepared qPCR mixtures were analyzed for RNA abundance using the CFX 96 Real-Time PCR Detection System (Bio-Rad) and normalized against the reference gene ribosomal protein S7 (AAEL009496, S1 Table). Collected data were analyzed using the Bio-Rad CFX Maestro software (Bio-Rad), and fold change values were calculated using the 2^-ΔΔCt method [42 (link)]. All reactions were run in duplicate (technical), and the gene expression differences reported were based on at least three independent experiments.