Thermal cycler dice real time system

Total RNA was extracted from the shrimp hemocytes prepared above using TRIzol^® reagent. 2 µg total RNA was used to the first-strand cDNA synthesis by random primers and M-MLV reverse transcriptase (Takara, Japan). Quantitative real-time RT-PCR was carried out using SYBR Premix Ex Taq™ (Takara, Japan) in a Thermal Cycler Dice^® Real Time System (Eppendorf, Germany) with the following conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 58°C for 10 s, and 72°C for 20 s. A dissociation curve with a single peak was used to monitoring the amplified product. 18S rRNA was used as the internal control. The primer sequences for the genes of four identified proteins and 18S rRNA were listed in Table 1. The data were calculated according to 2^−ΔΔCt method and statistically analyzed by SPSS 19.0.

Total RNA was extracted from different tissues of shrimp using TRIzol reagent (Invitrogen, USA), and then treated with RNase-free DNase I (TaKaRa, Japan). The first-strand cDNA was synthesized from 2 μg of DNA-free total RNA by M-MLV reverse transcriptase (Promega, USA) according to the manufacturer’s protocol. Quantitative real-time RT-PCR (qRT-PCR) was used to analyze the mRNA expression levels of FcSUMO and FcUBC9 in different tissues. The gene specific primers SUMO-F4 and SUMO-R4 were used to amplify a 150 bp product of FcSUMO, and specific primer pairs UBC9-F4 and UBC9-R4 were used to amplify a 121 bp product of FcUBC9, while the 18S rRNA primer pair 18S-F and 18S-R were used for amplification of the internal control fragment for qRT-PCR. PCR was carried out using SYBR Premix Ex Taq^™ (Takara) in a Thermal Cycler Dice^® Real Time System (Eppendorf, Germany) with the following conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 58°C for 10 s, and 72°C for 20 s. A dissociation curve with a single peak was used to monitoring the amplified product. The data were calculated according to 2^-ΔΔCt method.