Evaluation of Reference Genes for Transcriptional Profiling in Two Cockroach Models

Abstract

The German cockroach, Blattella germanica, and the American cockroach, Periplaneta americana are the most common and synanthropic household pests of interest to public health. While they have increasingly served as model systems in hemimetabolous insects for studying many biological issues, there is still a lack of stable reference gene evaluation for reliable quantitative real-time PCR (qPCR) outputs and functional genomics. Here, we evaluated the expression variation of common insect reference genes, including the historically used actin, across various tissues and developmental stages, and also under experimental treatment conditions in these two species by using three individual algorithms (geNorm, BestKeeper, and NormFinder) and a comprehensive program (RefFinder). RPL32 in B. germanica and EF1α in P. americana showed the overall lowest variation among all examined samples. Based on the stability rankings by RefFinder, the optimal but varied reference genes under specific conditions were selected for qPCR normalization. In addition, the combination of RPL32 and EF1α was recommended for all the tested tissues and stages in B. germanica, whereas the combination of multiple reference genes was unfavorable in P. americana. This study provides a condition-specific resource of reference gene selection for accurate gene expression profiling and facilitating functional genomics in these two important cockroaches.

RNAi of yellow‐y, required for normal cuticle pigmentation, impairs courtship behavior and oviposition in the German cockroach (Blattella germanica)

“…Five adults were set as one biological replicate and three replicates in total. Glyceraldehyde‐3‐phosphate dehydrogenase ( GAPDH , Genbank ID: WCD39345.1) and elongation factor‐1‐alpha ( EF1a , PSN34991.1) were used as the reference genes (He et al, 2021; Zhu et al, 2021).…”

Two Antenna-Enriched Carboxylesterases Mediate Olfactory Responses and Degradation of Ester Volatiles in the German Cockroach Blattella germanica

“…The mRNA levels were measured in five technical replicates using 2 × PerfectStar Green qPCR SuperMix (TransGen Biotech, Beijing, China), as described by the manufacturer. mRNA levels were quantified using three reference genes, glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), elongation factor-1-alpha ( EF1 α), and ribosomal protein L32 ( RPL32 ). , The 20 μL reaction volume contained 10 μL of qPCR SuperMix, 0.4 μL of forward/reverse primer (10 μM), and 2 μL of cDNA (final concentration of 17.5 ng/μL). qPCR was performed on an iQ5 Real-Time PCR system (Bio-Rad, CA, USA) using a program of 94 °C for 30 s followed by 50 cycles at 94 °C for 5 s and 60 °C for 30 s. All primer efficacies were nearly 100%, and the relative expression level of the mRNA was calculated according to the 2 –△△ Ct method using the formula expression levels = 2 − false[ ( Cq _ target − false( Cq _ ref false) ] prefix− [ Cq − control − false( Cq _ ef false) ] …”

Antimicrobial peptide expression in the cockroach gut during enterobacterial infection is specific and influenced by type III secretion

“…From CT values, expression of each AMP gene was calculated relative to the common housekeeping gene, EF1A ( Lin et al, 2014 ; Ray et al, 2020 ; Zhu et al, 2021 ), using the delta-CT method. In instances where expression of a particular AMP gene was not detectable (e.g.…”