qPCR was performed using the high-throughput platform BioMark™ HD System and the 48.48 GE Dynamic Arrays (Fluidigm) in duplicates in assays. 5 μL of Fluidigm sample premix consisted of 1 μL of either 20x or 40x diluted pre-amplified cDNA, 0.25 μL of 20x SG loading reagent (Fluidigm), 2.5 μL of Sso Fast Eva green mastermix (Bio-Rad), 0.1 μL of 4x diluted ROX (Invitrogen) and 1.15 μL of RNase/DNase-free water. Each 5 μL assay premix consisted of 2 μL of 10 μM primers (final concentration 400 nM primers), 2.5 μL 2x Assay loading reagent (Fluidigm) and 0.5 μL of RNase/DNase-free water. The samples and assays were mixed inside the chip using Nanoflex IFC controller (Fluidigm). Thermal conditions for qPCR were: 98°C for 40 s, 35 cycles of 95°C for 10 s, and 60°C for 40 s plus melting curve analysis. Data was processed by automatic threshold for each assay, with derivative baseline correction using BioMark Real-Time PCR Analysis Software 3.1.2 (Fluidigm). The quality threshold was set at the default setting of 0.65.
48.48 ge dynamic arrays
Manufactured by Standard BioTools
The 48.48 GE Dynamic Arrays are laboratory equipment designed for high-throughput genomic analysis. They enable the simultaneous processing of 48 samples against 48 assays, facilitating efficient and accurate data generation. The core function of these arrays is to enable parallel sample processing and analysis without further elaboration on their intended use.
Automatically generated - may contain errors
Lab products found in correlation
Biomark hd system, by Standard BioTools (2 mentions)
Assay loading reagent, by Standard BioTools (2 mentions)
Biomark real time pcr analysis software 3, by Standard BioTools (1 mentions)
Ssofast evagreen master mix, by Bio-Rad (1 mentions)
Nanoflex ifc controller, by Standard BioTools (1 mentions)
Ifc controller mx, by Standard BioTools (1 mentions)
Ge sample loading reagent, by Standard BioTools (1 mentions)
Taqman gene expression pcr master mix, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay 20, by Thermo Fisher Scientific (1 mentions)
2 protocols using 48.48 ge dynamic arrays
1
High-Throughput qPCR Using BioMark HD System
2
High-Throughput Quantitative PCR on Fluidigm Platform
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Quantitative PCR was performed using the high-throughput platform BioMark™ HD System and the 48.48 GE Dynamic Arrays (Fluidigm). 6 μL of Sample Master Mix (SMM) consisted of 2.7 μL of 5× diluted pre-amplified cDNA, 0.3 μL of 20× GE Sample Loading Reagent (Fluidigm) and 3 μL of TaqMan® Gene Expression PCR Master Mix (Life Technologies, Thermo Fisher). Each 6 μL Master Mix Assay (MMA) consisted of 3 μL of TaqMan® Gene Expression assay 20× (Life Technologies, Thermo Fisher) and 3 μL of 2× Assay Loading Reagent (Fluidigm). 5 μL of each SMM and each MMA premix were added to the dedicated wells. The samples and assays were mixed inside the chip using an MX IFC controller (Fluidigm). The parameters of the thermocycler were set with ROX as a passive reference and single probe FAM-MGB as a fluorescence detector.
Thermal conditions for qPCR were 25 °C for 30 min and 70 °C for 60 min for thermal mix; 50 °C for 2 min and 95 °C for 10 min for hot start; and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. To determine the quantification cycle Cq, data were processed with an automatic threshold for each assay, with linear derivative baseline correction using
BioMark Real-Time PCR Analysis Software 4.0.1 (Fluidigm). The quality threshold was set at a default setting of 0.65.
Thermal conditions for qPCR were 25 °C for 30 min and 70 °C for 60 min for thermal mix; 50 °C for 2 min and 95 °C for 10 min for hot start; and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. To determine the quantification cycle Cq, data were processed with an automatic threshold for each assay, with linear derivative baseline correction using
BioMark Real-Time PCR Analysis Software 4.0.1 (Fluidigm). The quality threshold was set at a default setting of 0.65.
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