High-throughput microfluidic fluorescence quantitative real-time PCR (qRT-PCR) was performed as previously described.15 Briefly, total RNA was extracted from MSCs using TRIzol (TaKaRa, Kusatsu, Japan) and then converted into cDNA using a PrimeScriptTM RT reagent kit (Takara). The sample pre-mix and assay pre-mix were prepared and added to the respective inlets of a 48.48 Dynamic Array integrated fluidic circuit (IFC, Fludigm, South San Francisco, CA, USA) that was primed and loaded in an IFC Controller MX (Fluidigm). qRT-PCR was performed in a BioMark HD System (Fluidigm), and the resulting data were analyzed using analysis software (Fluidigm). Real-time PCR was performed using a LightCycler 480 Real-Time PCR Detection System (Roche, Basel, Switzerland) using SYBR Green I Master Mix (Roche). The specific primers used for each gene are shown in Table 2 . Data were first normalized by glyceraldehyde-3-hosphate dehydrogenase (GAPDH) expression and then by the values in HDMSCs before LPS stimulation. The relative expression levels of each gene were determined using the 2−ΔΔCt method.
Ifc controller mx
Manufactured by Standard BioTools
Sourced in United States
The IFC Controller MX is a laboratory equipment product that serves as a central control unit for managing and coordinating various components in a microfluidic system. It provides the necessary functionality to operate and monitor the performance of connected devices, such as pumps, valves, and sensors, within a controlled environment. The IFC Controller MX is designed to facilitate the seamless integration and operation of microfluidic-based experiments and applications.
Automatically generated - may contain errors
Lab products found in correlation
Biomark hd system, by Standard BioTools (5 mentions)
Ge sample loading reagent, by Standard BioTools (2 mentions)
Lightcycler 480 real time pcr detection system, by Roche (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr green 1 master mix, by Roche (1 mentions)
Trizol, by Takara Bio (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
C1 preamp ifc, by Standard BioTools (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Bx x700, by Keyence (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Preamp master mix, by Standard BioTools (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
48.48 dynamic array, by Standard BioTools (1 mentions)
Real time pcr analysis software, by Standard BioTools (1 mentions)
Taqman gene expression assay 20, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression pcr master mix, by Thermo Fisher Scientific (1 mentions)
Assay loading reagent, by Standard BioTools (1 mentions)
48.48 ge dynamic arrays, by Standard BioTools (1 mentions)
6 protocols using ifc controller mx
1
High-throughput qRT-PCR Analysis of MSCs
2
Fluidigm 48.48 Real-Time PCR Protocol
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The Fluidigm 48.48 real-time PCR run was performed according to the manufacturer's instructions (Fluidigm, San Diego, CA, USA). Before performing real-time PCR, the sample mixture and assay mixture were prepared individually. The mixture for each sample (final volume 6 µL) contained 3 µL 2 × TaqMan® Universal PCR Master Mix (Applied Biosystems, PN 4304437), 0.3 µL 20×GE Sample Loading Reagent (Fluidigm, PN 85000746) and 2.7 µL diluted preamplification product as the template. The assay mixture contained 3 µL 2 × Assay Loading Reagent and 3 µL 20 × primer/probe mixture in a final volume of 6 µL. The final concentrations of primers and probes are shown in Table 2 . The samples and assay reagents were loaded into separate reaction chambers on the chip on an IFC controller MX (Fluidigm) after adding 5 µL mixture per assay inlet and per sample inlet. The array chip was run on a BioMark HD System (Fluidigm) using a protocol provided in the manufacturer's instructions. The protocol was as follows: a pre-digestion step of 50°C for 2 min; 95°C initial denaturation and UNG deactivation for 10 min; 50 cycles of 15 s at 95°C (denaturation) and 1 min at 60°C (annealing and extension). Fluorescence signals were monitored and analyzed at the annealing and extension steps during every PCR cycle using Q-PCR Analysis Software 3.0.2. (Fluidigm).
3
Single-cell gene expression analysis of cultured organoids
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Cultured organoids were dissociated into single cells using TrypLE select (Thermo Fisher Scientific, Waltham, MA, USA) during the third culture passage. Single-cell suspensions were loaded onto the C1 preamp IFC (10–17 μm, Fluidigm San Francisco, CA, USA), for single-cell isolation. Single-cell capture was confirmed under a microscope (BX-X700, Keyence, Osaka, Japan). Pre-amplification of target genes was performed using TaqMan assays (Thermo Fisher Scientific, Waltham, MA, USA), with the IFC controller MX (Fluidigm, San Francisco, CA, USA). Further amplification and data acquirement of the multiplex quantitative PCR analysis was done using the Biomark 48.48. dynamic array IFC and Biomark HD system (Fluidigm, San Francisco, CA, USA), according to manufacturer’s instructions. The set of target genes, and the corresponding gene-specific TaqMan assays analyzed in the study can be found in the Suppl. Table S2.
4
Gene Expression Profiling using Fluidigm Biomark
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Fluidigm 48.48 Biomark gene expression assays were performed, as described previously20 (link),40 (link). Briefly, the integrated fluidic chip (IFC) (Fluidigm) was primed using the IFC Controller MX, according to the manufacturer’s instructions. cDNA was diluted 1:1 with DEPC-treated water and 20× GE Sample Loading reagent was diluted 1:9 in TaqMan PCR Master Mix. Diluted cDNA and loading reagent were combined 1:1 and loaded onto the IFC chip. Primer assays were diluted 1:1 with 2X GE Assay Loading reagent and loaded into the IFC. The sample and assay were subsequently distributed on the chip via the IFC Controller MX loading program and the gene expression assay was performed and analysed using the GE 48.48 Standard.pcl on the Fluidim Biomark. Ct values were acquired from the Fluidigm Biomark, being normalised to ROX.
5
RNA Extraction and qPCR Analysis
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Cells were lysed in TRIzol LS reagents (Life technologies) and frozen at −80 °C for future use. Process of total RNA extraction was performed according to the manufacturer’s instructions. Reverse transcription to cDNA was performed with 1 μg using a High Capacity cDNA Archive Kit (Life technologies). cDNA samples were pre-amplified using the PreAmp master mix (Fluidigm) and the mixtures of all the Taqman assays (lifetechnologies) to prepare preamplified cDNA by PCR (18 cycles), following the manufacturer’s instruction. Pre-amplified samples were loaded in a 48.48 dynamic array integrated fluidic circuit (IFC; Fluidigm) using an MX IFC controller (Fluidigm). mRNA expression data were processed by BioMark HD System and data analyzed using the real-time PCR analysis software (Fluidigm). mRNA levels were normalized to Gapdh and data were analyzed by applying the 2^(-dCt) calculation method. A list of primers can be found in Supplementary Table 1 .
6
High-Throughput Quantitative PCR on Fluidigm Platform
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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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