Laboratory confirmation was performed following the protocol described by the Centers for Disease Control and Prevention (CDC) [14 ] for the confirmatory diagnosis of influenza A/H1Npdm09, A/H1N1, A/H3N2 and influenza B. Pharyngeal swabs were taken with Dacron swabs and preserved in viral transport medium (Becton Dickinson 7 Loveton Circle Sparks, USA, Cat: 220220) at 4 °C prior to use. To perform the virus determination, nucleic acids were extracted from 200 µL of the sample using the MagNa Pure LC Total Nucleic Acid Isolation Kit automated system (Roche Diagnostics, Mannheim, Germany; catalogue: 03038505001). The amplification was performed with the SuperScript III Platinum One-step qRT-PCR System (Invitrogen Carlsbad, California, EUA; cat: 12574035) in the 7500 Fast Real-Time PCR System (Applied Biosystems Foster City, California, EUA).
Superscript 3 platinum one step qrt pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperScript III Platinum One-step qRT-PCR System is a laboratory equipment product designed for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis. It provides a one-step solution for the detection and quantification of RNA targets.
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Lab products found in correlation
Exiprep dx viral dna rna kit, by Bioneer (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Magna pure lc total nucleic acid isolation kit, by Roche (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Chromo4 thermal cycler, by Bio-Rad (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rq1 rnase free dnase, by Promega (2 mentions)
Rneasy 96 kit, by Qiagen (1 mentions)
Platinum taq, by Thermo Fisher Scientific (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rna mini kit, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Rneasy mini spin column, by Qiagen (1 mentions)
Balb c mice, by Charles River Laboratories (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
35 protocols using superscript 3 platinum one step qrt pcr system
1
Influenza Virus Diagnostic Protocol
2
Quantitative RT-PCR for Gene Knockdown
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Total RNA was harvested from cells using RNeasy 96 kit (Qiagen) for measuring KD efficiency upon shRNA transduction. Cellular RNAs were reverse transcribed, and PCR amplified using the SuperScript III Platinum One-Step qRT-PCR System with Platinum Taq (Invitrogen) and IDT Primer Assays (Integrated DNA Technologies). Cellular RNAs were normalized to 18S levels using StepOnePlus System (Applied Biosystems).
3
Quantification of Ribonuclease P Expression
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To evaluate the presence and quality of the purified RNA, the constitutive expression of the ribonuclease P (RNase P) gene was measured. For this, specific primers, and probes (F-AGATTTGGACCTGCGAGCG; R-GAGCGGCTGTCTCCACAAGT; and probe HEX-TTCTGACCTGAAGGCTCTGCGCG-BHQ1) [17 (link)], were prepared at a concentration of 10 µM. Amplification was performed using the SuperScript™ III Platinum™ One-Step qRT-PCR System (Invitrogen, cat: 11732-088; Lot: 2223827; Invitrogen Life Technologies. Carlsbad. CA 92008. USA), following the manufacturer's recommendations. Of this mixture, 20 µL was placed in each well. Subsequently, 5 µL of the purified RNA was added, obtaining a final volume of 25 µL. The following controls were included in the plate: NTC, mix Vero-76 cells DENV-1- and DENV-2-infected (positive control), and mock-infected (negative control). The plate was amplified in the QuantStudio™ 5 thermocycler (Thermo Applied Biosystems, cat: A28134) and the amplification was performed with the following thermal profile: 15 min at 50°C, 2 min at 95°C, 15 sec at 94°C, and finally 30 sec at 58°C, the last two repeating 42 cycles.
4
Quantitative PCR of GLI1 and PTCH1
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RNA was extracted using RNA mini kit (Thermo Scientific). Primers and TaqMan probes for detection of human Tata binding protein (TBP), GLI1, and PTCH1 were purchased as Assays-on-Demand from Applied Biosystems (TBP, Hs00427620_m1; GLI1, Hs01110766_m1; PTCH1, Hs00181117_m1). SuperScript III Platinum One-Step qRT-PCR System (Invitrogen) was used for the qPCR (PCR protocol: 15 min 50°C, 2 min 95°C, 30–50× 15 s 95°C and 1 min 60°C). The amount of amplicon generated during the PCR was measured using a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Each sample was run in triplicate; controls without reverse transcriptase gave no signal in all samples.
5
Bat DPP4 Expression Profiling
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To study DPP4 expression profiles in different bat cell lines, cell lysates were collected for total RNA extraction using RNeasy Mini Spin Column (QIAgen). RNA was eluted in 50 μl of RNase-free water and was used as a template for one-step RT-qPCR with SuperScript III platinum One-step qRT-PCR system (Invitrogen, Carlsbad, CA, USA). RT-qPCR assays were performed using conserved primers designed by multiple alignment of available bat DPP4 gene sequences (Table 2 ), using β-actin for normalization. cDNA was amplified in a LightCycler 480 (Roche, Basel, Switzerland) with 25 μl reaction mixtures containing 2 × reaction mix, 5 μl RNA, 25 μM ROX reference dye, 50 μM primers, and 10 μM probe at 50 °C for 30 min, then 95 °C for 2 min followed by 50 cycles of denaturation, annealing and extension. Experiments were performed in triplicates, and results were expressed as the mean expression level of DPP4/β-actin. The relative expression between different bat cells was then calculated by the ΔΔCt method.
6
Influenza A Infection in BALB/c Mice
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First, 6- to 8-week-old female BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) were anesthetized with isoflurane and intranasally inoculated with PBS or PBS-diluted viruses (102–106 TCID50 of each virus) in 20 µL PBS. The mice were weighed and monitored daily for clinical signs of infection. Animals with prostration signs or with severe weight loss (≥25%) were euthanized, and their tissues were harvested and frozen at −80 °C. The thawed tissues from the 105 TCID50-infected mice were weighed and homogenized in 0.5 mL PBS using a Tissuelyser II (Qiagen, Hilden, Germany), and the viral titers were determined by TCID50 analysis. The viral RNAs from sera were extracted using a QIAmp viral RNA mini kit (Qiagen). Real-time RT-PCR was performed with 5 µL of the sample using a SuperScript III Platinum one-step qRT-PCR system (Invitrogen, Carlsbad, CA, USA) in Mastercycler Realplex 2 (Eppendorf, Hamburg, Germany) with appropriate primers for the detection of influenza A and M segment [12 (link)].
7
Quantitative RT-PCR Analysis of Viral RNA
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RNA was extracted from the cell supernatant using the Maxwell 16 equipment with the Maxwell 16 Total RNA purification kit (Promega, Madison, WI, USA), following the manufacturer’s instructions.
The reverse transcription technique followed by PCR was used to obtain the number of viral copies. We used the Superscript III Platinum One-Step qRT-PCR System commercial kit with Rox separated, according to the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). The RT-PCRs were performed on the ABI PRISM 7500 standard (Applied Biosystems, Waltham, MA, USA).
The reactions of all the viruses investigated comprised a final reaction of 25 μL, containing 12.5 μL of 2× reaction mix with Rox, 5.5 μL of nuclease-free water, 1.0 μL of SLEV/BREV F/R primer mix, 0.5 μL of SLEV/BREV probe, 0.5 μL of SIII/TaqPI enzyme mix, and 5.0 μL of total RNA, using the following cycling conditions: 1 × 45 °C cycle for one h and 94 °C for 3 min; 40 × 94 °C cycles for 30 s, 55 °C for one min; and 68 °C for 3 min. The respective primers and probes highlighted inTable 1 were used.
The reverse transcription technique followed by PCR was used to obtain the number of viral copies. We used the Superscript III Platinum One-Step qRT-PCR System commercial kit with Rox separated, according to the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). The RT-PCRs were performed on the ABI PRISM 7500 standard (Applied Biosystems, Waltham, MA, USA).
The reactions of all the viruses investigated comprised a final reaction of 25 μL, containing 12.5 μL of 2× reaction mix with Rox, 5.5 μL of nuclease-free water, 1.0 μL of SLEV/BREV F/R primer mix, 0.5 μL of SLEV/BREV probe, 0.5 μL of SIII/TaqPI enzyme mix, and 5.0 μL of total RNA, using the following cycling conditions: 1 × 45 °C cycle for one h and 94 °C for 3 min; 40 × 94 °C cycles for 30 s, 55 °C for one min; and 68 °C for 3 min. The respective primers and probes highlighted in
8
Quantitative Detection of Influenza Virus
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To detect infectious virus, previously frozen ferret nasal wash specimens were titered by standard plaque assay in MDCK cells47 . To detect and quantify viral RNA copy numbers, total RNA was extracted from samples using the RNeasy Mini Kit (Qiagen). Real-time RT-PCR was performed with a SuperScript III Platinum One-Step qRT-PCR System (Invitrogen) in duplicate reactions using an influenza A virus M1 gene primer and probe set (Influenza A Forward Primer: GAC CRA TCC TGT CAC CTC TGA C; Influenza A Reverse Primer: AGG GCA TTY TGG ACA AAK CGT CTA; Influenza A Probe FAM—TG CAG TCC TCG CTC ACT GGG CAC G—BHQ)48 . Influenza virus M gene RNA copy numbers were extrapolated using a standard curve based on samples of known M gene copy number.
9
Dengue and JEV Detection Protocol
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Dengue virus and JEV infections were detected using Panbio ELISAs (PanBio Ltd., Sinnamon Park, Queensland, Australia), i) the JEV-Dengue IgM Combo ELISA (Cat. No. E-JED01C) for the detection of anti-dengue and anti-JEV IgM, ii) Dengue Early ELISA (Cat. No. E-DEN01P) for the detection of dengue NS1 antigen, iii) and Dengue IgG capture ELISA (Cat. No. E-DEN02G) for the detection of high level anti-dengue IgG associated with acute secondary infection. Primary and secondary dengue infections were distinguished using the Dengue IgG indirect ELISA (Cat. No. E-DEN01G) on admission serum when the IgG capture was ELISA negative.
RNA was extracted from sera (140μl) using QIAamp Viral RNA kit (Qiagen) following manufacturer’s instructions in 80μl elution volume. Dengue TaqMan real-time RT-PCR was performed with the SuperScriptIII Platinum One-Step qRT-PCR system (Invitrogen), with 5μl of RNA extract in a 25μl reaction volume [45 (link)]. Pan-dengue RT-PCR was performed on all samples and positive samples were submitted to the four specific RT-PCRs for serotyping.
RNA was extracted from sera (140μl) using QIAamp Viral RNA kit (Qiagen) following manufacturer’s instructions in 80μl elution volume. Dengue TaqMan real-time RT-PCR was performed with the SuperScriptIII Platinum One-Step qRT-PCR system (Invitrogen), with 5μl of RNA extract in a 25μl reaction volume [45 (link)]. Pan-dengue RT-PCR was performed on all samples and positive samples were submitted to the four specific RT-PCRs for serotyping.
10
Cytokine expression analysis in Hec1b cells
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For those strains that promoted morphological alterations in Hec1b cells were performed analysis of the cytokines (IL10 and TNFα) production. Total RNA was extracted using the Trizol Reagent (Invitrogen, Calsbag, CA, USA). RNA yield was estimated by Nanodrop (Thermo Scientific). A minimum amount of 0.2 ng of the RNA was submitted to reverse transcription followed by qRT PCR. Real-time PCR primers are listed in Table 2 and the reaction was performed using the StepONE Plus thermocycler (Applied Biosystems). Each 10 μL reaction contained 400 nM of each primer, 5 μL Master Mix and 1/60 000 Fast EVA Green Master Mix (both from Biotium), 0.25 uL of Super Script III Platinum One-Step qRT-PCR System (Invitrogen Calsbad, CA, USA) and the recommendations for cDNA production described in the kit were followed. Reactions were repeated on three different biological replicates using as endogenous control the GAPDH gene and fold expression changes were averaged. The data was analyzed by the comparative Ct method revised in (Schmittgen and Livak 2008 (link)).
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