RNA was extracted from the supernatants of virally infected cells using either TRIzol reagent (15596; Life Technologies, Inc.) or a Purelink Pro 96 viral RNA/DNA kit (12280; Invitrogen). SuperScript III (18080; Invitrogen) was used to synthesize cDNA using random hexamers. Quantitative PCR was performed on a 7500 Fast real-time PCR system (Applied Biosystems). SuperScript III RT/Platinum Taq (Thermo 2574030) was used with the primers 5′-GACCRATCCTGTCACCTCTGAC-3′ and 5′-AGGGCATTYTGGACAAAKCGTCTA-3′, and the TaqMan probe 6-carboxyfluorescein (FAM)-TGCAGTCCTCGCTCACTGGGCACG-3′ with Blackhole Quencher 1 with an annealing temperature of 55°C for M segment copy number measurement. Quantification of cDNA copy number based on threshold cycle (CT) values was performed using standard curves from 10-fold dilutions of plasmid containing the M gene of A/Puerto Rico/8/1934 H1N1. The ratio of the infectious titer per milliliter to the genome copy number per milliliter is the specific infectivity of the sample.
Superscript 3 rt platinum taq
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperScript III RT/Platinum Taq is a combined reverse transcription and PCR amplification system. It features a reverse transcriptase enzyme and a DNA polymerase enzyme for efficient conversion of RNA to cDNA and subsequent DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Purelink pro 96 viral rna dna kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cellsdirect 2x reaction mix, by Thermo Fisher Scientific (1 mentions)
Biomark hd system, by Standard BioTools (1 mentions)
Bst 2.0 dna polymerase, by New England Biolabs (1 mentions)
Synthetic dna, by Thermo Fisher Scientific (1 mentions)
Rnaseout recombinant ribonuclease inhibitor, by Thermo Fisher Scientific (1 mentions)
Hx ifc controller, by Standard BioTools (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
96.96 dynamic array, by Standard BioTools (1 mentions)
Taqman assays, by Standard BioTools (1 mentions)
Biomark hd, by Standard BioTools (1 mentions)
5 protocols using superscript 3 rt platinum taq
1
Quantifying Viral Genome Copies and Infectivity
2
Single-cell transcriptomics of islet macrophages
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Islet macrophages were isolated, stained, and single-cell-sorted into 96-well plates containing 5 μl Cellsdirect 2X Reaction Mix (Thermo Fisher Scientific) and 0.1 μl RNase inhibitor (SUPERase-In; Thermo Fisher Scientific) per well. For reverse transcription and specific cDNA preamplification, all the 96 20× Taqman assays (Fig. S5; IDT DNA) were mixed and diluted to 0.2×. Subsequently, 2.5 μl of the 0.2× Taqman assay mix, 0.2 μl of the SuperScript III RT/Platinum Taq (Thermo Fisher Scientific), and 1.2 μl PCR-certified water (Thermo Fisher Scientific) were added together to the sorted single cells. Reverse transcription (15 min at 50°C) was performed, followed by Taq activation (2 min at 95°C) and 18 cycles of cDNA preamplification (each cycle: 15 s at 95°C, 4 min at 60°C). The resulting cDNA product was diluted with PCR-certified water (1:5) and submitted for single-cell quantitative PCR using the Biomark HD system (Fluidigm).
3
pH-LAMP Protocol for HPV DNA and mRNA Detection
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The pH-LAMP mix for HPV DNA test contained the following: 2.4 L of Betaine (stock at 5 M), 1.5 L of customised isothermal buffer (pH 8.5–9), 0.9 L of MgSO4 (stock at 100 mmol/L), 0.9 L of bovine serum albumin (20 mg/mL), 0.84 L of dNTPs (stock at 25 mmol/L), 0.375 L of Syto9 (20 mmol/L stock), 0.375 L of sodium hydroxide (0.2 M), 0.04 L of Bst 2.0 DNApolymerase (120,000 U/mL) (New England Biolabs, Hitchin, UK), 1.5 L of primer mixture (20 mmol/L BIP/FIP, 10 mmol/L LB/LF, and 2.5 mmol/L B3/F3), 3 µL of synthetic DNA (Integrated DNA Technologies) template solution, and enough nuclease-free water to bring the volume to 15 L. The pH-LAMP mix for hTERT and GAPDH mRNA tests contained the same reagents as the HPV DNA test, in addition to 0.375 L of SuperScript III RT/Platinum Taq (Thermo FisherScientific, Waltham, USA), 0.15 L of Recombinant Ribonuclease Inhibitor (Thermo FisherScientific, Waltham, USA) and enough nuclease-free water to bring the volume to 15 L.
4
Single-cell RT-PCR Analysis with Fluidigm
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Single-cell RT-PCR were performed using 96:96 IFC microfluidic chips (Fluidigm). Single cells were flow sorted directly into the individual wells of 96-well PCR plates containing 5 μl/well of CellsDirect PCR mix and RNase Inhibitor mix. Following single-cell sorting, each well was supplemented with 1 μl SuperScript III RT/Platinum Taq (Invitrogen), 1.5 μl Tris-EDTA (TE) buffer and 2.5 μl of 96 pooled TaqMan assays (Applied Biosystems). Single-cell lysates were directly reverse transcribed into cDNA (50 °C for 15 min, 95 °C for 2 min) and pre-amplified for 14 PCR cycles (60 °C for 4 min, 95 °C for 15 s). A mixture containing 2.25 μl amplified cDNA (diluted 1:5), 2.5 μl TaqMan quantitative PCR (qPCR) mix (Applied Biosystems) and 0.25 μl Fluidigm sample loading agent was prepared and loaded onto the IFC chip. A 2.5 μl aliquot of TaqMan assay was mixed with 2.5 μl Fluidigm assay loading agent and loaded onto the IFC chip. The 96:96 chip was then submitted to HX IFC Controller (Fluidigm) before setting up qPCR run on Biomark real-time PCR machine (Fluidigm) following the manufacturer’s instructions; the data were analyzed using the Fluidigm program. Cells not expressing ACTB (β-actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were removed from the analysis.
5
Single-Cell Gene Expression Profiling
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Single-cell gene-expression experiments were performed using 96.96 Dynamic Array (Fluidigm Inc.). Single cells were sorted into individual wells of 96-well PCR plates and preloaded with 5 µl/well of CellsDirect One-Step qRT-PCR mix (Thermo Fisher); then each well was supplemented with 1 µl of SuperScript-III RT/Platinum Taq (Invitrogen) and a mixture of 96 pooled TaqMan assays (Fluidigm Inc., 5 µM each). Single-cell mRNA was reverse transcribed (50°C for 15 min, 95°C for 2 min) and pre-amplified for 22 cycles (95°C for 15 s, 60°C for 4 min). Libraries and probes were transferred into 96.96 Dynamic Array integrated fluidic circuits (IFCs) for qRT-PCR on a BioMark HD instrument (both Fluidigm Inc.) following the manufacturer’s instructions.
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