Selection and Validation of Potential Reference Genes for Quantitative Real-Time PCR Analysis in Blaptica Dubia (Serville, 1838) (Blattidae, Blaberidae)
Abstract
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an effective, reproducible, and dependable method for evaluating and targeting expression of genes. It is very important to normalize according to stably expressed housekeeping genes in order to facilitating gene expression studies and to acquire exact and meaningful results. The purpose of this study was to identify and validate six housekeeping genes (GADPH, RPS18, α-TUB, EF1α, ArgK and ACTB) in adults of cockroach species Blaptica dubia employing five different algorithms (geNorm, Bestkeeper, Normfinder, ΔCt method and RefFinder) to assess putative housekeeping gene expression stability. Our study also showed that the geNorm, Normfinder ΔCt method and RefFinder algorithms identified GADPH as the most stable housekeeping gene in B. dubia adults. Additioanlly, RPS18 was suggested as the most stable gene by GeNorm and BestKeeeper. ACTB has been shown to be by far the least stable of all algorithms. In addition, since there are few validation studies for reference genes in cockroaches in the literature, it is considered that it would be beneficial to increase the number of studies related with RT-qPCR on the reference genes validation under biotic and abiotic conditions in cockroaches.
Keywords
Blaptica dubia, RT-qPCR, Reference genes, Validation, Normalization.
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