Real-time PCR was performed using the QuantStudio™ 3 real-time PCR System instrument (Applied Biosystems, Foster City, CA, USA) as described previously (Ishii et al., 2012 (link); James et al., 2015 (link); Mehta et al., 2019 (link); Mehta and Singh, 2017 ). A set of representative genes of NCCs were selected to define the cells in this study (Ishii et al., 2012 (link); Szabo and Mayor, 2018 (link); Trainor, 2005 (link)) (Table 1 ). All data are representative of three experimental set of cells with PCR triplicates and are presented as the fold change.
Quantstudio 3 real time pcr system instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio™ 3 real-time PCR System is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It is capable of quantifying and detecting target nucleic acid sequences in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ave buffer, by Qiagen (1 mentions)
Universal sybr green fast qpcr mix, by ABclonal (1 mentions)
Lunascript rt supermix kit, by New England Biolabs (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using quantstudio 3 real time pcr system instrument
1
Real-Time PCR Analysis of Neural Crest Cells
2
RNA Extraction and qPCR Quantification
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RNA was extracted from 140 μL of cell supernatants with QIAamp Viral RNA Mini Kit (QIAGEN, # 52906) and eluted in 60 μL of buffer AVE (QIAGEN). RNA ligations were diluted tenfold and used for reverse transcription without extraction. The reverse-transcription (RT) reactions were performed with 2X LunaScript® RT SuperMix Kit (NEB) with 5 μL of RNA input in 10 μL total volume. The RT reactions containing cDNA were diluted 100-fold and 5 μL was used for qPCR reactions with 2X Universal SYBR Green Fast qPCR Mix (ABClonal). Each qPCR reaction contained 5 uL of cDNA, 4.2 μL of Nuclease-free Water, 0.4 μL of 10 mM Primers (Table S4 ), and 10 μL of 2X mastermix. Nuclease-free water was used as negative template control (NTC). Three technical replicates were performed for each sample. Amplification was performed in QuantStudio 3 Real-Time PCR System instrument (Applied Biosystems) as follows: 95°C for 3 min, 40 cycles of 95°C for 5 s and 60°C for 30 s, followed by Melt Curve analysis. Results were analyzed in Design and Analysis app at Thermo Fisher Connect Platform.
3
RNA Extraction and qRT-PCR Analysis
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RNA extraction and qRT-PCR methods were conducted as previously published [53 (link)]. In brief, the total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and the RNA was converted into cDNA using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Furthermore, using cDNA as a template, RT-PCR analysis was performed using the Applied Biosystems Quant Studio 3 Real-Time PCR System instrument. The primers used in this assay are listed in Supplementary Table S1 .
4
Woodchuck Hepatic IFN Expression
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Changes in the intrahepatic expression of IFN-α, IFN-β, and IFN-γ were determined by real-time PCR, as described in more detail in the Supplementary Data . In brief, total RNA was isolated from liver tissue using the RNeasy Mini kit (Qiagen, Redwood City, CA, USA) with on-column digestion by RNase-free DNase I (Qiagen). Following reverse transcription of mRNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) using oligo(dT), complementary (c) DNA samples were amplified on a QuantStudio 3 Real Time PCR System instrument (Applied Biosystems) with the TaqMan Gene Expression Master mix (Applied Biosystems) and woodchuck-specific primers and probes (Table S1 ). Woodchuck 18S rRNA expression was used to normalize IFN expression. IFN transcription levels were calculated as a fold-change relative to the pretreatment baseline at week −1 using the formula 2ΔCt.
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