Topscript one step rt pcr drymix

To detect SFTSV, nested PCR was performed using MF3 and MR2 primers to target the M fragment (Table 2). Reactions were performed in a total volume of 20 μL, containing TOPscript One-step RT PCR DryMix (Enzynomics, Republic of Korea), 1 μL of each primer (10 pmol/μL), 5 μL of template RNA, and 13 μL of distilled water (DW). The reaction conditions comprised cDNA synthesis steps at 50 °C for 30 min and at 95 °C for 10 min. Following this, 35 cycles of 95 °C for 20 s, 58 °C for 40 s, and 72 °C for 30 s were performed, followed by a final extension at 72 °C for 5 min. For the second amplification, reactions were performed in a total volume of 20 μL, containing 10 μL of 2× TOPsimple DyeMix (aliquot)-HOT (Enzynomics, Republic of Korea), 1 μL of each primer (10 pmol/μL), 1 μL of ^1stPCR product, and 7 μL of DW, using MMF3 and MMF2 primers. Reaction conditions comprised a denaturation step at 94 °C for 5 min, followed by 35 cycles of 94 °C for 20 s, 59 °C for 20 s, and 72 °C for 20 s. After this, a final extension was performed at 72 °C for 5 min.

RT-PCR was performed using TOPscript One-step RT PCR Drymix (Enzynomics, Daejeon, Korea), according to the manufacturer’s instructions. The primer sets used were as follows: hP2Y₂R, 5′-GTG CTC TAC TTC CTG GCT-3′ and 5′-CTG AAG TGT TCT GCT CCT AC-3′ and hGAPDH, 5′- TCA ACA GCG ACA CCC ACT CC-3′ and 5′- TGA GGT CCA CCA CCC TGT TG-3′.

Recombinant human TNF-α protein and human IL-1β/IL-1F2 Quantikine ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). ATP, apyrase, anti-β-actin (#A2066) antibody, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-NLRP3 (#ab214185), anti-NLRC4 (#ab99860), and anti-cleaved caspase-1 (#ab179515) antibodies and human VEGF-A ELISA kits were purchased from Abcam (Cambridge, United Kingdom), and anti-ASC (#13833) antibody was obtained from Cell Signaling Technology (Beverly, MA, USA). TOPscript One-step RT PCR Drymix was purchased from Enzynomics (Daejeon, Korea). Negative control siRNA (control siRNA) and NLRP3, NLRC4, ASC, caspase-1, and P2Y₂R siRNA were obtained from Bioneer (Daejeon, Korea), and Lipofectamine 3000 reagent, TRIzol reagent, and anti-P2Y₂R (#PA1-46150) antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hybond-P⁺ polyvinylidene difluoride membrane was obtained from GE Healthcare (Chicago, IL, USA), and Clarity Western enhanced chemiluminescence (ECL) substrate was purchased from Bio-Rad (Hercules, CA, USA). BD Matrigel basement membrane matrix (Matrigel) was obtained from BD Bioscience (Franklin Lakes, NJ, USA), and cell culture inserts for 24-well plates (8.0 µm, Translucent PET membrane) were purchased from Corning (Corning, NY, USA).

Total RNA was extracted using TRIzol Reagent (Invitrogen), and RT-PCR was performed using the TOPscript One-step RT PCR DryMIX (Enzynomics, Daejeon, Korea) according to the manufacturer's instructions. The primer sets used were as follows: hP2Y₂R, 5′-GTG CTC TAC TTC CTG GCT-3′ and 5′-CTG AAG TGT TCT GCT CCT AC-3′ and hGAPDH, 5′- TCA ACA GCG ACA CCC ACT CC-3′ and 5′- TGA GGT CCA CCA CCC TGT TG-3′. Thirty cycles of amplification were performed under the following conditions: melting at 95°C for 30 sec, annealing at 56°C for 30 sec, and extension at 72°C for 30 sec.

Total RNA was extracted using TRIzol reagent (15596018, Thermo Fisher Scientific, Rockford, IL, USA). All primers were obtained from Bioneer, and RT-PCR was performed using TOPscript One-step RT-PCR Drymix (RT421, Enzynomics, Daejeon, Korea) according to the manufacturer’s instructions. The primer sets were as follows: hA1R, forward 5′-TCCCTCTCCGGTACAAGATG-3′ and reverse 5′-GCTGCTTGCGGATTAGGTAG-3; hA2AR, forward 5′-AGCTGAAGCAGATGGAGAGC-3′ and reverse 5′-AGGGATTCACAACCGAATTG-3; hA2BR, forward 5′-CAGCGGGAGATCCATGCAG-3′ and reverse 5′-CGGTTCCGGTAAGCATAGACAAT-3; hA3R, forward 5′-TACCCACGCCTCCATCATGT-3′ and reverse 5′-GGGGTCAATCCCACCAGGA-3; hCD39, forward 5′-CTGATTCCTGGGAGCACAT-3′ and reverse 5′-GACATAGGTGGAGTGGGAGAG-3; hCD73, forward 5′-GCCTGGGAGCTTACGATTTTG-3′ and reverse 5′-TAGTGCCCTGGTACTGGTCG-3; hGAPDH, forward 5′-TCAACAGCGACACCCACTCC-3′ and reverse 5′-TGAGGTCCACCACCCTGTTG-3. Thirty cycles of amplification were performed under the following conditions: melting at 95 °C for 30 s, annealing 57.5 or 60 °C for 30 s, and extension at 72 °C for 1 min.

Total RNA was extracted from breast cancer cells (1 × 10⁶ cells) using TRIzol reagent (15596018, Thermo Fisher Scientific, Waltham. MA, USA) according to the manufacturer’s protocol. RT–PCR was conducted using TOPscript One-step RT–PCR Drymix (RT421, Enzynomics, Daejeon, Korea) by the manufacturer’s instructions. Human ESM-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were purchased from Bioneer (Deajeon, Korea), and the sequences of these primers were as follows: hESM-1 forward, 5′-GC CCT TCC TTG GTA GGT AGC-3′, and reverse, 5′-TG TTT CCT ATG CCC CAG AAC-3′, and hGAPDH forward, 5′- TCA ACA GCG ACA CCC ACT CC-3′, and reverse, 5′-TGA GGT CCA CCC TGT TG-3′. Amplification was conducted under the following conditions: 27 cycles of denaturation at 95 °C for 30 sec, annealing at 57.5 °C for 30 sec, and extension at 72 °C for 1 min.