Stratagene mx3000 real time pcr system

The mRNA expression profiles of SMPD in different tissues and at different developmental stages and the relationship of SMPD with the reproductive status of workers and queens were examined by real-time quantitative PCR. Total RNA was extracted, and first-strand cDNA was generated using the PrimeScript First-strand cDNA Synthesis kit (Takara Bio Inc., Dalian, China) following the manufacturer’s instructions. Real-time PCR was performed using the SYBR Premix ExTaq II kit (Takara, Dalian, China) and a Stratagene Mx3000 real-time PCR system (Agilent, United States). The primers SMPDF-F and SMPDF-R were designed based on the full-length sequence of SMPD obtained from the RACE-PCR analysis. The expression of β-actin and GAPDH transcripts, which were used as endogenous controls, was assessed using the primers β-actin-F/R and GAPDH-F/R (Table 1). The amplification conditions were as follows: 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Each sample was replicated three times, and relative SMPD expression levels were quantified using the comparative 2^−ΔΔCt method (Livak and Schmittgen 2001 (link)). One-way analysis of variance (ANOVA) was applied using R-Project software (version 3.3.2) to assess the differences in expression level between different reproductive statuses (P < 0.05).