CMV particles were added to a premix containing reverse transcriptase, primers, and a probe with master mix containing DNA polymerase, dNTP, buffer (Tris-HCl, KCl, MgCl2, pH8.5), etc. The reverse transcriptase and master mix used were RocketScript™ reverse transcriptase, RNase H minus (Bioneer), and QuantStudio™ 3D Digital PCR Master Mix v2 (Thermo Fisher Scientific), respectively. The prepared reaction solution containing CMV particles was injected into a loading blade, loaded onto a digital PCR chip (QuantStudio™ 3D Digital PCR 20 K Chip Kit v2, Thermo Fisher Scientific) comprising sub-nanoliter-sized chambers using a QuantStudio™ 3D Digital PCR Chip Loader (Thermo Fisher Scientific) and sealed.
Rocketscript reverse transcriptase
Manufactured by Bioneer
RocketScript™ is a reverse transcriptase enzyme used in the synthesis of complementary DNA (cDNA) from RNA templates. It facilitates the conversion of RNA into single-stranded cDNA, a crucial step in various molecular biology applications such as gene expression analysis, cDNA library construction, and reverse transcription-PCR (RT-PCR).
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d digital pcr 20k chip kit v2, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital pcr master mix v2, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital pcr chip loader, by Thermo Fisher Scientific (1 mentions)
Topscript cdna synthesis kit, by Enzynomics (1 mentions)
Nuclease free water, by Merck Group (1 mentions)
Nanodrop uv spectrophotometry, by Thermo Fisher Scientific (1 mentions)
3 protocols using rocketscript reverse transcriptase
1
Digital PCR Detection of CMV Particles
2
Viral RNA to cDNA Synthesis Protocols
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cDNA was synthesized from purified viral RNA or RNA in a heat-degraded leaf sample using two different commercial kits at room temperature. At 50°C, The TOPscript cDNA synthesis kit (Enzynomics Daejeon, South Korea) was used, while the RocketScript™ Reverse transcriptase (Bioneer, Daejeon, South Korea) was used at 70°C. Synthesis procedures were carried out according to manufacturer protocols.
3
Droplet Digital PCR for RNA Quantification
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For ddPCR experiment, the sample mixtures were prepared in a 50 µL of master mix (one-step RocketScript™ Reverse Transcriptase, Bioneer, Korea) containing 1 µL of the template RNA, 1 µL of primers (10 µM each) and 1 µL of probes (10 µM) following the manufacturer’s protocol. Highly concentrated samples were diluted in nuclease-free water (Sigma-Aldrich, USA). The initial concentration was quantified by Nanodrop UV spectrophotometry (NanoDrop 2,000, Thermo Scientific, USA). The samples were partitioned by a droplet-based microfluidic chip as previously reported in our research group [26 ]. The droplets were collected in a tube and isothermal ddPCR was conducted at 39 °C for 20 min. The resulting droplets were pipetted onto slide glass and the fluorescence images were taken by a fluorescence microscopy (IX73, Olympus, Japan).
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