Applied biosystems 7500 7500 fast real time system

We used the qRT-PCR to test PmC1qDC-1 expression levels. The mix reagent was from DyNAmo Flash SYBR Green qPCR kit (ThermoFisher Scientific). The qRT-PCR experiment of PmC1qDC-1 was carried out on Applied Biosystems 7500/7500 Fast Real-Time System (Applied Biosystems, Foster City, CA, USA). The PCR program was as follows: 95 °C for 5 min, 40 cycles at 95 °C for 30 s, 60 °C for 15 s, 72 °C for 15 s. The relative expression levels of reference genes (β-actin and GAPDH) and PmC1qDC-1 were calculated through the 2^−ΔCT method.
Significance was analyzed using SPSS 22.0 (IBM, Chicago, IL, USA). The expression levels of PmC1qDC-1 at the tissues, different development stages, and different time points of shell notching were analyzed using one-way ANOVA. Differences in PmC1qDC-1 expression between the two groups were evaluated using the T-test. The significant level for these analyses was set at P < 0.05.

Total RNA was prepared using Trizol reagent and then treated with DNase I (Promega) to eliminate genomic DNA contamination. Templates for mRNA amplification were prepared using oligo (dT)-adaptor primers and specific primer with M-MLV reverse transcriptase (Promega, USA). Considering the genomic and transcriptome data from our laboratory (data not shown), we obtained partial sequences of PmRunt and PmCBF in P. martensii. The 5′ and 3′ ends of PmRunt cDNAs were obtained by rapidly amplifying the cDNA ends.
qRT-PCR assay was performed to quantify the expression of target genes. Thermo Scientific DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific) was used according to the manufacturer’s protocol. Fluorescence was detected in Applied Biosystems 7500/7500 Fast Real-time system (Applied Biosystems, Foster City, CA, USA). The relative expression levels of the target genes were calculated through the 2^−ΔΔct method, and U6 or GAPDH were used as reference gene. All PCR primers used in this study are listed in S1 Table.