Ribomax large scale rna production systems sp6 and t7 kit

The primers for PCR amplification of the target sequence are shown in Table 1. The dsRNA was synthesized using a RiboMAX™ Large Scale RNA Production Systems-SP6 and T7 kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions and purified with the Trizol Isolation Reagent (Invitrogen, Waltham, MA, USA). An RNA interference assay was carried out with double-strand RNA (dsRNA) transfection into Hpt cell cultures. Hpt cells (5 × 10⁵ cells) were seeded into 24-well culture plates and cultured in 500 μL of L15 medium (HyClone, Logan, UT, USA) with minor modifications. Four hundred nanograms of dsRNA was transfected with 0.8 μL of Cellfectin II Reagent (Invitrogen, Waltham, MA, USA) in Hpt cells according to the manufacturer’s protocol. After 36 h, Hpt cells were infected with WSSV (MOI = 2) and collected at 12 hpi for RNA extraction. The dsRNA-targeted GFP was used as negative control.

IVT-mRNAs were synthesized from pGE-Core, pGE-SP-Core, and pGE-GFP vectors (Fig. 1B, 1C, and 1D) after the linearization of the constructs with HindIII and GsuI restriction enzymes (MBI Fermentas, St Leon-Rot, Germany). The IVT-mRNAs synthesis was performed using RiboMAX™ Large-Scale RNA Production Systems-SP6 and T7 Kit (Promega Corporation, Madison, USA) according to the manufacturer’s instruction. DNA templates were purified using a PCR purification kit and used for in vitro transcription. Transcription of 5-10 µg of the DNA templates was carried out in a final 20–200 µl reaction mixture at 37 °C for 4 hours in order to generate uncapped IVT- mRNA. To remove the template DNA, 1 μl of RQ1 RNase-Free DNase (Catalog Numbers: M6101) was added to the IVT reaction mixture and incubated at 37°C for 15 min. Purification of mRNA was performed after DNaseI digestion by Total RNA Purification Kit (Jena-Bioscience , Germany), according to the instructions provided by manufacturer. The purity and quality of the RNAs were determined using spectrophotometric analysis and run on the agarose gel (1.1%). The mRNAs were stored in the RNase-free water at -80 °C.