Gene expression and expressed SNVs were assessed by qPCR or SNP type PCR across single cells using a Biomark HD system (Fluidigm). cDNAs obtained from the C1 array for mRNA sequencing chip were subjected to specific target amplification following the manufacturer’s recommendations. For the gene expression qPCR, Delta Gene Assay (Fluidigm) with EvaGreen second generation dsDNA binding dye was performed for gene sets selected from the RS genes (Additional file 6 ). To compare correlations between RNA-seq and qPCR platforms for the selected 43 gene expression, mean fold change over median expression was calculated as in the previous study [33 (link)]. Validation of expressed SNVs at the RNA level was carried out using a SNP Type Assay (Fluidigm) with locus-specific primer sequences. Primers were designed using D3™ software (Fluidigm), and sequences are available in Additional file 6 .
Delta gene assay
Manufactured by Standard BioTools
Sourced in United States
The Delta Gene Assay is a nucleic acid-based assay designed for gene expression analysis. The core function of the Delta Gene Assay is to quantify and compare the expression levels of target genes across multiple samples.
Automatically generated - may contain errors
Lab products found in correlation
D3 software, by Standard BioTools (2 mentions)
Snptype assay, by Standard BioTools (1 mentions)
Biomark hd system, by Standard BioTools (1 mentions)
Te buffer, by Teknova (1 mentions)
Reverse transcription master mix, by Standard BioTools (1 mentions)
Preamp master mix, by Standard BioTools (1 mentions)
Fluidigm real time pcr analysis software, by Standard BioTools (1 mentions)
Exonuclease 1, by New England Biolabs (1 mentions)
Biomark hd, by Standard BioTools (1 mentions)
Qubit assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
5 protocols using delta gene assay
1
Single-cell gene expression and SNV analysis
2
Comparative qPCR and RNA-seq Analysis
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qPCR was performed with the DELTAgene assay (PN100-3035, Fluidigm) using cDNAs from 6 bulk and 185 single-cell samples. Primer sequences were designed using D3 software (Fluidigm) and are listed in Supplementary Table 6 . Before comparison of qPCR and RNA-seq data, Ct values of 999 (=not detected) were replaced with ‘NA'. Ct values were negatively converted and −20 was set as the threshold value. These data represent the log2 expression level for qPCR comparable to log2(TPM+1) for RNA-seq. The inter-relations were assessed by Pearson's correlation, Spearman's rank order correlation and linear regression analysis.
3
Quantitative Gene Expression Analysis
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250 ng of total RNA was used to generate cDNA for each sample using Reverse Transcription Master Mix (Fluidigm, San Francisco, CA). The cDNA was pre-amplified for 14 cycles with the primers of DELTAgene Assay (Fluidigm, San Francisco, CA) in a multiplex PCR reaction using PreAmp Master Mix (Fluidigm, San Francisco, CA). The pre-amplified PCR products were treated with Exonuclease I (New England Biolabs, Ipswich, MA). The pre-amplified Exonuclease treated products was diluted 5-fold using TE buffer (TEKnova, Hollister, CA) and used as sample in the Sample inlets of primed Dynamic Array 96.96 GE IFC (Fluidigm, San Francisco, CA). 5 μM concentration of each primer of DELTAgene Assay was added to Assay inlets of primed Dynamic Array 96.96 GE IFC, according to manufacturer’s instruction. The real-time PCR data was collected using the Biomark HD (Fluidigm, San Francisco, CA) with the instrument settings as specified by the manufacturer. Each sample had 4 replicates. The fold change of gene expression was calculated using Fluidigm Real-Time PCR Analysis software (Fluidigm, San Francisco, CA).
4
Comprehensive MDSC Gene Expression Analysis
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A customised 48-gene panel (Delta Gene Assay, Fluidigm, California, USA) was designed, based on phenotypic and functional markers related to MDSC as reported in the literature from in vitro or in vivo studies of various diseases in humans or animal models (Table 1
Controls included no-template controls, no-reverse-transcription controls, and no-pre-amplification controls. Upon successful completion of the Fluidigm assay the Ct-values (the fractional number of cycles required for the signal to cross the detection threshold), proportional to the inverse gene expression levels, were exported for further analysis.
Controls included no-template controls, no-reverse-transcription controls, and no-pre-amplification controls. Upon successful completion of the Fluidigm assay the Ct-values (the fractional number of cycles required for the signal to cross the detection threshold), proportional to the inverse gene expression levels, were exported for further analysis.
5
Quantitative PCR of PDZ Genes
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Total RNA was extracted using a Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) and quantified using the Qubit™ assay kit and a Qubit 2.0 Fluorometer (Life Technologies, Waltham, MA, USA).
For expression analysis, 64 PDZ genes were selected from PDZ genes with high and medium expression levels in diverse immune cells as previously reported [46 (link)]. The gene set was completed with 11 PDZ genes belonging to the MAGUK subfamily and 8 PDZ genes involved in trafficking, signaling, and recycling processes. Five reference genes were included in the relative expression analyses. Primers were designed using a Deltagene assay (Fluidigm, South San Francisco, CA, USA) and are listed inSupplementary Table S9 .
Total RNA (200 ng of total RNA in a 20 μL volume reaction) was used to synthesize cDNA using the OneStep RT-PCR Kit (Qiagen, Hilden, Germany). A pool of the 88 primer pairs (Supplementary Table S9 ) was used under the following conditions: 50 °C for 30 min, 95 °C for 15 min, followed by 15 cycles of pre-PCR cycling amplification at 94 °C for 30 s, 60 °C for 60 s, and 72 °C for 60 s. The pre-amplified product was diluted 15-fold with Tris/Ethylenediaminetetraacetic acid (TE) Buffer.
For expression analysis, 64 PDZ genes were selected from PDZ genes with high and medium expression levels in diverse immune cells as previously reported [46 (link)]. The gene set was completed with 11 PDZ genes belonging to the MAGUK subfamily and 8 PDZ genes involved in trafficking, signaling, and recycling processes. Five reference genes were included in the relative expression analyses. Primers were designed using a Deltagene assay (Fluidigm, South San Francisco, CA, USA) and are listed in
Total RNA (200 ng of total RNA in a 20 μL volume reaction) was used to synthesize cDNA using the OneStep RT-PCR Kit (Qiagen, Hilden, Germany). A pool of the 88 primer pairs (
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