Briefly, 96-well plates were prepared with 5 × 105 cells/mL of Vero E6 (200 µL per well), as previously described [10 (link)]. The different concentrations of methylene blue without photoactivation, hydroxychloroquine or remdesivir were added 4 h before infection. The replication of IHUMI-3 or IHUMI-6 strains in Vero E6 cells at an MOI of 0.01 was estimated 48 h after infection by RT-PCR using the Superscript III platinum one step with Rox kit (Invitrogen) after extraction with the BioExtract SuperBall kit (Biosellal, Dardilly, France). The primers used were previously described [33 (link)]. EC50 (median effective concentration) and EC90 (90% effective concentration) were estimated through nonlinear regression by using the R software (ICEstimator version 1.2). EC50 and EC90 values resulted in the mean of 6 to 12 independent experimentations.
Superscript 3 platinum
Manufactured by Thermo Fisher Scientific
Sourced in United States
Superscript III Platinum is a reverse transcriptase enzyme used for the synthesis of first-strand cDNA from RNA templates. It provides high thermal stability and increased specificity for RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Faststart universal sybr green master, by Roche (1 mentions)
Fast sybr green, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Abi 7900ht system, by Thermo Fisher Scientific (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
6 protocols using superscript 3 platinum
1
Antiviral Testing of Methylene Blue, Hydroxychloroquine, and Remdesivir
2
Single-cell RT-PCR for Spinal Neuron Analysis
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Single-cell RT-PCR for spinal neurons was performed as previously described61 (link). Briefly, the contents of dissociated DRG neurons were harvested into patch pipettes with tip diameters of about 20 μm, gently put into reaction tubes containing Dnase I, then were kept at 37 °C for 40 min and followed by 80 °C for 10 min to remove contamination of genomic DNA. After adding reverse transcriptase (SuperScript III Platinum, Invitrogen), the samples were mixed gently and incubated at 50 °C for 50 min. The reaction was stopped by heating sample to 70 °C for 15 min. The cDNA products were used in gene-specific nested PCR. The sequences of the primers are shown in Table 1 . The first round PCR was carried out using FastStart Universal SYBR Green Master (Roche, Switzerland). The following PCR conditions were used: 1 cycle of 3 min, 94 °C; 35 cycles of 15 s, 95 °C; 15 s, 60 °C; 1 cycle of 10 min, 72 °C. The second round of PCR was performed using 0.5 μl of the first PCR product as the template. The amplification regents and procedure for the inner primers was the same as that of the first round. A negative control was obtained from pipettes that did not have cell contents but were submerged in the bath solution. The PCR products were displayed on GelRed-stained agarose gels (3%).
3
Quantitative RT-PCR Assay Protocol
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cDNA synthesis and qPCR (RT-qPCR) assays were carried out using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). Reactions were performed with the SuperScript® III Platinum® (Invitrogen) with SYBR Green Fast (Applied Biosystems). Reactions contained 50 ng of total RNA and 600 nM of each primer. PCR conditions were 50 °C for 15 min followed by 95 °C for 2 min, 45 cycles of 95 °C for 15 s and 60 °C for 1min. Primer pair specificity was evaluated by melting curve analysis and gel electrophoresis. Assays were performed with technical triplicates.
4
Real-time RT-PCR for AIBV Detection
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A one step Real time RT-PCR was carried out using Invitrogen kit (SuperScript® III Platinum, Life Technologies, USA). The synthesis of the cDNA first strand was performed using 5 μl total viral RNA primed with a universal pair of primers a) downstream primer, AIBV-fr, targeting N gene nucleotide positions 811–832 (5′-ATGCTCAACCTTGTCCCTAGCA-3′); and b) upstream primer, AIBV-as, targeting N gene nucleotide positions 921–941 (5′-TCAA-ACTGCGGATCA-TCACGT-3′), and the probe TaqMan targeting N gene nucleotide positions 848-875 (5′FAM-TTGGAAGTAGAGTGACGCCCAAACTTCA-3′Tamra) [31 (link)].
The amplifications reactions (PCRs) were performed on a Smart Cycler. The following mix of each reaction was contained: 12.5 μl 2 × RT- PCR buffer mix, 0.5 μl MgSO4 (50 mM), 0.5 μl Rox (25 mM), 4.75 μl nuclease free water, 0.5 μl M-MULV reverse transcriptase enzyme (200U), 0.5 μl primers to a final concentration of 10 μM, 0.25 μl probe to a final concentration of 10 μM and 5 μl RNA template. The reaction was carried out in StepOneTM Plus real-time PCR system (Smart cycler Cepheid, USA) at 50 °C for 15 min, 95 °C for 5 min, and 40 cycles of 95 °C for 15 s and 60 °C for 45 s. All reactions amplifications were recorded, analyzed, and the threshold cycle (Ct) determined with the StepOne software (Smart Cycler).
The amplifications reactions (PCRs) were performed on a Smart Cycler. The following mix of each reaction was contained: 12.5 μl 2 × RT- PCR buffer mix, 0.5 μl MgSO4 (50 mM), 0.5 μl Rox (25 mM), 4.75 μl nuclease free water, 0.5 μl M-MULV reverse transcriptase enzyme (200U), 0.5 μl primers to a final concentration of 10 μM, 0.25 μl probe to a final concentration of 10 μM and 5 μl RNA template. The reaction was carried out in StepOneTM Plus real-time PCR system (Smart cycler Cepheid, USA) at 50 °C for 15 min, 95 °C for 5 min, and 40 cycles of 95 °C for 15 s and 60 °C for 45 s. All reactions amplifications were recorded, analyzed, and the threshold cycle (Ct) determined with the StepOne software (Smart Cycler).
5
Quantifying Gene Expression via qRT-PCR
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Total RNA was extracted using the Directzol RNA miniprep kit (Zymo Research) and analyzed by Taqman-based qRT-PCR (SuperScript III Platinum; Life Technologies) on an ABI 7900HT system (Applied Biosystems). Samples were analyzed as multiplexed reactions with 36B4 as internal control. Quantification was carried out using the standard curve method.
6
Confirming Virus Identity and Infectivity
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Prior to inoculation of experimental animals, all stored isolates were screened by qPCR to detect SPV genomes to confirm the virus identity and then revived in diploid cells to confirm the presence of infectious virus after long term storage.
A one step Real time PCR was carried out using Invitrogen kit (SuperScript® III Platinum, Life Technologies, USA). The DNA amplification was performed using universal pair of primers a) downstream primer, CAPV-074F1, targeting gene encoding to envelop protein P32 (5′AAA ACG GTA TAT GGA ATA GAG TTG GAA-3′); and b) upstream primer, CAPV-074R1, 5′-AAA TGA AAC CAA TGG ATG GGA TA-3′, and the Taqman probe: 5′-FAM-TGG CTC ATA GAT TTC CT-MGBNFQ-3′ (12 (link)). The amplifications reactions (PCRs) were performed on a Smart Cycler. The following mix of each reaction was contained: 12.5 μl 2× PCR buffer mix, 0.5 μl MgSO4 (50 mM), 0.5 μl Rox (25 mM), 4.75 μl nuclease free water, 0.5 μl primers to a final concentration of 10 μM, 0.25 μl probe to a final concentration of 10 μM and 5 μl DNA template. The reaction was carried out in Step One TM Plus real-time PCR system (Smart cycler Cepheid, USA) at 94°C for 5 min, 94°C for 10 min, and 40 cycles of 55°C for 15s and 72°C for 45s. All reactions amplifications were recorded, analyzed, and the threshold cycle (Ct) determined with the Step One software (Smart Cycler).
A one step Real time PCR was carried out using Invitrogen kit (SuperScript® III Platinum, Life Technologies, USA). The DNA amplification was performed using universal pair of primers a) downstream primer, CAPV-074F1, targeting gene encoding to envelop protein P32 (5′AAA ACG GTA TAT GGA ATA GAG TTG GAA-3′); and b) upstream primer, CAPV-074R1, 5′-AAA TGA AAC CAA TGG ATG GGA TA-3′, and the Taqman probe: 5′-FAM-TGG CTC ATA GAT TTC CT-MGBNFQ-3′ (12 (link)). The amplifications reactions (PCRs) were performed on a Smart Cycler. The following mix of each reaction was contained: 12.5 μl 2× PCR buffer mix, 0.5 μl MgSO4 (50 mM), 0.5 μl Rox (25 mM), 4.75 μl nuclease free water, 0.5 μl primers to a final concentration of 10 μM, 0.25 μl probe to a final concentration of 10 μM and 5 μl DNA template. The reaction was carried out in Step One TM Plus real-time PCR system (Smart cycler Cepheid, USA) at 94°C for 5 min, 94°C for 10 min, and 40 cycles of 55°C for 15s and 72°C for 45s. All reactions amplifications were recorded, analyzed, and the threshold cycle (Ct) determined with the Step One software (Smart Cycler).
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