A total of 2 μL of RNA was reverse transcribed into cDNA using RT reagent (Fluidigm, San Francisco, CA, USA), using a programmed thermal cycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: 5 min at 25 °C; 30 min at 42 °C; 5 min at 85 °C; and finally held at 4 °C. Pre-amplification of TaqMan probes (all ThermoFisher Scientific Waltham, MA, USA, Supplementary Table S2 ) and cDNA was carried out as previously described [32 (link)]. Transcript analysis was performed by qPCR using the Biomark HD gene expression system (Fluidigm, San Francisco, CA, USA), as previously described [18 (link)]. Hierarchal clustering of the data was performed using Cluster 3.0 and Java TreeView v.1.1.6 [31 (link)]. Genes showing a high degree of clustering were further analysed by normalizing values to the respective samples’ own undifferentiated MSCs using the ∆∆Ct method [37 (link)]. The use of this methodology allowed for patient variability to be accounted for in the downstream statistical analysis by assisting in the normalizing of values.
Biomark hd gene expression system
Manufactured by Standard BioTools
Sourced in United States
The Biomark HD gene expression system by Standard BioTools is a high-throughput digital PCR platform designed for accurate and sensitive gene expression analysis. The system integrates microfluidic technology to enable parallel processing of multiple samples and targets, providing efficient and comprehensive data generation.
Automatically generated - may contain errors
Lab products found in correlation
Reverse transcription kit, by Standard BioTools (2 mentions)
Preamp master mix, by Standard BioTools (2 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Quantstudio design and analysis software, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5, by Thermo Fisher Scientific (1 mentions)
Microamp optical 384 well reaction plate, by Thermo Fisher Scientific (1 mentions)
Microamp optical 96 well reaction plate, by Thermo Fisher Scientific (1 mentions)
Quantstudio 7, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
3 protocols using biomark hd gene expression system
1
Reverse Transcription and qPCR Analysis
2
T-cell Transcriptome Analysis Protocol
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T-cells were sorted using an Influx (BD Biosciences, San Jose, CA, USA) cell sorter directly into RNA extraction buffer, supplied as a component of the PicoPure RNA isolation kit (Thermofisher Scientific, Waltham, MA, USA). cDNA was synthesized with a reverse transcription kit (Fluidigm, San Francisco, CA, USA) and then underwent pre-amplification (18 cycles) using a pre-amp master mix (Fluidigm, San Francisco, CA, USA) with a solution containing all primer sets. All primers were purchased from Applied Biosystems. Transcript analysis was performed by qPCR using the Biomark HD gene expression system (Fluidigm, San Francisco, CA, USA), and values displayed are the log 10ΔCt relative to the Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) housekeeping gene (Supplementary Table S2 ).
3
Single-Cell Transcriptional Profiling of T Cells
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T cells were sorted using an Influx (BD) cell sorter directly into RNA extraction buffer, supplied as a component of the PicoPure RNA isolation kit (Thermo Fisher). cDNA was synthesised with a reverse transcription kit (Fluidigm) then underwent preamplification (18 cycles) using a pre-amp master mix (Fluidigm) with a solution containing all primer sets. All primers were purchased from Applied Biosystems. Transcript analysis was performed by qPCR using the Biomark HD gene expression system (Fluidigm); values displayed are log 10ΔCt relative to the HPRT1 housekeeping gene.
qRT-PCR was completed using the TaqMan Gene expression assay as per the manufacturer’s instructions (Applied Biosystems). Samples, no template control (NTC) and positive controls (pooled cDNA) were loaded onto either a MicroAmp Optical 96-Well Reaction Plate (Applied Biosystems) or a MicroAmp Optical 384-Well Reaction Plate (Applied Biosystems) and run on either the Quantstudio 5 or Quantstudio 7 instrument, respectively (both from Applied Biosystems) using the Quantstudio Design and Analysis software (V. 1.4, Thermo Scientific), the Comparative Threshold (Ct) value was subsequently normalised using the HPRT1 housekeeping gene and unstimulated control for 2-ΔΔCt. All primers were purchased from Applied Biosystems. A full list of primers used can be found inonline supplementary table 2 .
qRT-PCR was completed using the TaqMan Gene expression assay as per the manufacturer’s instructions (Applied Biosystems). Samples, no template control (NTC) and positive controls (pooled cDNA) were loaded onto either a MicroAmp Optical 96-Well Reaction Plate (Applied Biosystems) or a MicroAmp Optical 384-Well Reaction Plate (Applied Biosystems) and run on either the Quantstudio 5 or Quantstudio 7 instrument, respectively (both from Applied Biosystems) using the Quantstudio Design and Analysis software (V. 1.4, Thermo Scientific), the Comparative Threshold (Ct) value was subsequently normalised using the HPRT1 housekeeping gene and unstimulated control for 2-ΔΔCt. All primers were purchased from Applied Biosystems. A full list of primers used can be found in
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