Primerscript rt reagent kit with gdna eraser perfect real time

Twelve DEGs in the transcriptome were selected for verification using qRT-qPCR to confirm the dependability of the transcriptome results. The same samples were used for both BGISEQ-500 sequencing and qRT-PCR validation. Total RNA of the antennal glands was extracted using the TRIzol reagent (Invitrogen, Carlsbad, US). The quantity and quality of RNA were determined by the NanoDrop 2000 micro-volume spectrophotometry (Thermo Fisher Scientific, Waltham, US) and the agarose gel electrophoresis, respectively. The cDNA was synthesized using the template of 2 μg extracted RNA by the PrimerScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara Biotechnology, Kusatsu, Japan). RT-qPCR was carried out in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, US) following the program of 95 °C for 30 s, 32 cycles of 94 °C for 5 s, 55 °C for 30 s, and 72 °C for 10 s. All samples were run in triplicate, and each assay was repeated three times. For the standard curves of each gene, the amplification efficiencies were 95–105 % and the coefficient (R²) were 0.98 < R². The relative gene expression was calculated using the 2^−ΔΔCq method as described by Schmittgen and Livak [60 (link)], where the β-actin of S. paramamosain was used as the reference gene.