Biomark™ HD with a 96.96 IFC was used for the RT-qPCR amplification, as previously described [17 (link)]. Briefly, for each sample, a 6 µL sample mix containing 3 µL of 2× SsoFast EvaGreen Supermix with low ROX (Bio-Rad Laboratories, Hercules, CA, USA), 0.3 µL of 20× DNA Binding Dye (Fluidigm), and 2.7 µL of the pre-amplified sample was prepared. A primer stock (100 μM combined forward and reverse primers) was prepared for each assay, and 0.3 µL of the stock was mixed with 3 µL of 2× Assay Loading Reagent (Fluidigm) and 2.7 µL 1× DNA Suspension Buffer to make assay mixes. Finally, 5 μL of each assay and sample mix was transferred into the appropriate inlets according to Fluidigm’s recommendation. After loading, the array was placed in the Biomark HD instrument for quantification and detection using the GE Fast 96 × 96 PCR + Melt v2.pcl PCR thermal protocol. The data were analyzed with Real-Time PCR Analysis Software (Fluidigm) according to Fluidigm’s recommendation. A non-template cDNA/pre-amplification and a non-template pre-amplification control (H2O) were included and were finally defined as those with Ct values ≥ 35 or that were undetermined.
Dna binding dye
Manufactured by Standard BioTools
Sourced in United States
The 20× DNA Binding Dye is a concentrated solution designed for staining and visualizing DNA in various applications, such as gel electrophoresis and fluorescence microscopy. It binds to DNA and emits a fluorescent signal when excited by a light source, allowing researchers to detect and analyze DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Assay loading reagent, by Standard BioTools (4 mentions)
Ssofast evagreen supermix with low rox, by Bio-Rad (3 mentions)
Biomark 96.96 ifc, by Standard BioTools (2 mentions)
Juno controller, by Standard BioTools (2 mentions)
Biomark hd qpcr platform, by Standard BioTools (2 mentions)
Real time pcr analysis software, by Standard BioTools (1 mentions)
Biomark real time pcr analysis software, by Standard BioTools (1 mentions)
Biomark system, by Standard BioTools (1 mentions)
Evagreen 2 qpcr mastermix, by Applied Biological Materials (1 mentions)
Rt pcr master mix, by Vazyme (1 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Cell culture flasks and dishes, by Sarstedt (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pcr certified water, by Teknova (1 mentions)
Dna suspension buffer, by Teknova (1 mentions)
Exonuclease 1, by New England Biolabs (1 mentions)
Te buffer, by Teknova (1 mentions)
Pcr tubes, by Sarstedt (1 mentions)
5 protocols using dna binding dye
1
Biomark HD qPCR Protocol for Gene Expression
2
Single-cell qPCR Analysis of Gene Expression
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Ninety-six individual primer sets were pooled to a final concentration of 0.1 μM for each primer as described29 (link). After 7 days of culture, 96 single cells were randomly picked from cultures incubated with control or the combination of CHIR-99021, Forskolin and OAC1 conditioned medium and sorted into 8-well PCR strips loaded with 5 μL RT-PCR Master Mix (Vazyme) in each well. Sorted strips were immediately frozen at –80 °C and immediately placed into the PCR machine after brief centrifugation. The PCR progress was identical to the multi-cell one-step PCR but with 20 cycles of sequence-specific amplification. After pre-amplification, PCR strips were stored at –80 °C to avoid evaporation. Pre-amplified products were diluted by 5-fold and analyzed with EvaGreen 2 × qPCR MasterMix (Applied Biological Materials, Vancouver, Canada), 20 × DNA Binding Dye (Fluidigm, San Francisco, CA, US) and individual qPCR primers using 96.96 Dynamic Arrays on a BioMark System (Fluidigm). Threshold crossing (Ct) values were calculated using the BioMark Real-Time PCR Analysis software (Fluidigm).
3
Characterization of CuO Nanoparticles
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All chemicals (p.a. grade), cell culture medium, and supplements were obtained either from Carl Roth GmbH (Karlsruhe, Germany) or Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) with the exception of fetal bovine serum (FBS), which was bought at Thermo Fisher Scientific GmbH (Dreieich, Germany). Cell culture dishes and flasks, reaction tubes, PCR tubes, and other consumables were purchased from Sarstedt (Nuembrecht, Germany). CuO NP were kindly provided by Dr. Wendel Wohlleben (BASF SE, Germany) in the course of the BMBF-funded project MetalSafety and originally synthesized and obtained from by PlasmaChem (Berlin, Germany). This particle species was also previously examined in the EU FP7-Project SUN, including oral and inhalation in vivo studies [28 (link),60 (link)]. Primer pairs were synthesized by Eurofins (Ebersberg, Germany). DNA suspension buffer, PCR certified water, and TE buffer were obtained from Teknova (Hollister, USA). The 2× Assay Loading Reagent, 20× DNA Binding Dye, and IFCs were purchased from Fluidigm (San Francisco, USA). The 2× SsoFast EvaGreen Supermix was provided by Bio-Rad (Munich, Germany), and the 2× TaqMan PreAmp Master Mix was bought from Applied Biosystems (Darmstadt, Germany). Exonuclease I was obtained from New England Biolabs (Frankfurt am Main, Germany).
4
High-throughput qPCR Analysis of Gene Expression
5
Highly Sensitive qPCR Profiling of Gene Expression
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The qPCR reaction mixture had a volume of 5 µL and contained 2.25 µL of diluted preamplified cDNA, 0.25 µL of DNA Binding Dye (Fluidigm), and 2.5 µL SsoFast EvaGreen Supermix with low ROX (Bio‐Rad). The primer reaction mixture had a final volume of 5 µL and contained 2.5 µL Assay Loading Reagent (Fluidigm) and 0.25 µL of a mix of all reverse and forward primers, corresponding to a final concentration of 500 nM in the reaction. The Biomark 96.96 IFC™ (Integrated Fluidic Circuit) was first primed with an oil solution in the Juno™ Controller (Fluidigm) to fill the fluidic circuit. In all, 96 sample reactions (5 µL each) were loaded into individual sample wells, and 96 forward and reverse primer mixtures were loaded into each assay wells (5 µL each). The IFC was then placed in the Juno™ Controller for automatic loading and mixing. After 90 minutes, the IFC was then transferred to the Biomark™ HD qPCR platform (Fluidigm). The cycling program consisted of Thermal Mix at 70°C for 40 minutes followed by 60°C for 30 seconds. Hot Start was 1 minutes at 95°C, followed by 30 cycles of denaturation at 96°C for 5 seconds, and annealing at 60°C for 20 seconds. Melting curves were collected between 60°C and 95°C with 1°C increments/3 seconds.
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