Topscript rt pcr kit

Total RNA was extracted from isolated SMG and HSG cells using the Hybrid-RiboEx extraction system (Gentaur, Belgium) following the manufacturer's instructions, and amplified according to the manufacturer's protocol using TOPscript™ RT-PCR kit from Enzynomics (Daejeon, South Korea) and nested primers. The primers used are listed in Table 1. The PCR protocol comprised a denaturation step at 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, an annealing step for 1 min, and an extension step at 72°C for 1 min, finally culminating with a final extension step at 72°C for 10 min. PCR products were electrophoresed on 1% agarose gels. Bands on agarose gels were visualized and acquired with a CCD camera and scanned using GelDoc^XR imaging system (Bio-Rad, CA). Intensities of PCR bands were analyzed with a MetaMorph system (Molecular Devices).

JEV GI RNA extraction was performed using the AccuPrep^® Viral RNA Extraction Kit (Bioneer, Daejeon, Republic of Korea). Complementary DNA (cDNA) was synthesized using the TOPscript™ RT-PCR Kit (Enzynomics, Daejeon, Republic of Korea) with a random hexamer. JEV GIII NS4B (GenBank: EF623989.1, nucleotides 6912 to 7649) and GV NS1 genes (GenBank: JF915894.1, nucleotides 2481 to 3542) were synthesized by Macrogen (Seoul, Republic of Korea). PCR of JEV GI was conducted using the prepared cDNAs and PCR of GIII and GV performed using the synthesized plasmid gene. Primers containing the T7 promoter sequence and Phusion^® High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs, MA, USA; Table S1) were employed. JEV RNAs were synthesized via IVT using PCR products and the mMESSAGE mMACHINE T7 Transcription kit (Invitrogen, MA, USA). Synthesized RNAs were purified using a GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Scientific™, Waltham, MA, USA).

Total RNA was extracted from the mouse cardiac tissues using the Hybrid-Ribo^Ex extraction system (301-001, GeneAll, Seoul, Korea) in accordance with the instructions of the manufacturer. Isolated RNA was quantitated using the Spectrophotometer ND-1000 (Thermo Fisher Scientific), and cDNA was amplified using the TOPscript RT-PCR kit from Enzynomics (RT410S, Daejeon, Korea). The primers and PCR cycling protocol were as follows: mouse Bcl-2 (forward) GCA TCT TCT CCT TCC AGC CTG and (reverse) GAC GCT CTC CAC ACA CAT GAC; mouse p53 (forward) CAT GGC CAT CTA CAA GAA GTC and (reverse) GAA CAT CTC GAA GCG TTT ACG; denaturation at 95 °C (5 min), followed by 35 cycles of 95 °C (1 min), an annealing step (1 min), and an extension step at 72 °C (1 min). The final extension step was performed at 72 °C (10 min).