Messenger RNAs for mouse genes involved in BA synthesis and hepatic BA transporters were quantified using the QuantiGene Plex 2.0 assay (Panomics/Affymetrix, Fremont, CA). Individual bead-based oligonucleotide probe sets were designed by Panomics/Affymetrix, Inc. using previously published NCBI gene accession numbers (panel IDs: 21140, 21065, and 21150). Assays were performed following the manufacturer’s protocol (Panomics/Affymetrix, Fremont, CA). Briefly, isolated RNA (250 ng) was hybridized overnight at 54 °C to specific oligonucleotide sequences, corresponding to the genes of interest that were bound to capture beads. A linearity test was done prior to the analysis. The following day the hybridized RNA, oligos, and capture beads were pipetted onto a filter plate. The complex was then sequentially hybridized to the pre-amplifier, followed by the amplifier, and lastly the biotinylated label probe (each for 1 h at 50 °C on a shaking platform). Streptavidin–phycoerythrin was then allowed to bind for 30 min at room temperature on a shaking platform. Fluorescence was detected and analyzed with a Bioplex 200 system array reader with Luminex xMAP technology (Bio-Rad, Hercules, CA). The housekeeper gene, Gapdh, was used to normalize the data. The data are expressed as a ratio of the relative light units of the target gene mRNA relative to Gapdh mRNA.
Quantigene plex 2.0 assay
Manufactured by Panomics
Sourced in United States
The Quantigene Plex 2.0 assay is a multiplex gene expression analysis platform. It enables simultaneous quantification of multiple target genes from a single sample.
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9 protocols using quantigene plex 2.0 assay
1
Quantification of Bile Acid Metabolism mRNAs
2
Quantification of Calprotectin and Type I IFN
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Levels of calprotectin (S100A8/A9) were analysed by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, Minnesota, USA). Type I IFN activity was measured using a reporter cell system as described previously.12 (link) Briefly, WISH epithelial cells were cultured with patient serum and analysed for induction of six IFN-regulated genes and three housekeeping genes using the Quantigene Plex 2.0 assay according to the manufacturer’s instructions (Panomics, Fremont, California, USA).
Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep according to the manufacturer’s protocol (Axis-Shield PoC, Oslo, Norway). Within the PBMCs, LDGs were identified as CD14+CD15+CD16++ cells with high forward and side scatter properties using flow cytometry.
Levels of ICs were assessed using IC-FLOW. Briefly, neutrophils were incubated with sera, diluted 1:10 and analysed for cell surface FcγRIIA availability by flow cytometry. In the presence of ICs, FcγRIIA is internalised and no signal is detected by flow cytometry, resulting in an inverse correlation between levels of cell surface FcγRIIA and levels of ICs. As a positive control, heat-aggregated IgG of known concentration was used.
Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep according to the manufacturer’s protocol (Axis-Shield PoC, Oslo, Norway). Within the PBMCs, LDGs were identified as CD14+CD15+CD16++ cells with high forward and side scatter properties using flow cytometry.
Levels of ICs were assessed using IC-FLOW. Briefly, neutrophils were incubated with sera, diluted 1:10 and analysed for cell surface FcγRIIA availability by flow cytometry. In the presence of ICs, FcγRIIA is internalised and no signal is detected by flow cytometry, resulting in an inverse correlation between levels of cell surface FcγRIIA and levels of ICs. As a positive control, heat-aggregated IgG of known concentration was used.
3
Quantifying Cytokine mRNA in Mouse Brains
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QuantiGene Plex 2.0 Assay (Panomics, Santa Clara, CA, United States) was used for cytokine mRNA quantification in the mouse brains as described elsewhere (Kottilil et al., 2009 (link)).
4
QuantiGene Plex 2.0 Assay Protocol
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The QuantiGene Plex 2.0 assay (Panomics/Affymetrix, Fremont, CA, USA) was used to confirm the microarray analysis. The assay was performed according to the manufacturer's instructions, accessible at https://tools.thermofisher.com/content/sfs/manuals/QuantiGene_Plex_User_manual.pdf . Briefly, tissue-extracted RNAs were added to a 96-well plate along with QuantiGene hybridization solution. The sealed hybridization plate was shaken at 54°C, 600 rpm overnight (18-22 h). The hybridization mixture was transferred to a prewetted filter plate, filtered, and washed three times in wash buffer. The samples were then incubated in three successive working solutions. Beads were identified, and fluorescent signals were measured. The raw data file contained signal intensity and background level for each gene in each sample.
5
Multiplex Gene Expression Quantification
6
Type I IFN Activity Measurement
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Type I IFN activity was measured as previously described assessing the capacity of circulating type I IFNs to signal through IFNAR [19 (link)–21 (link)]. Briefly, WISH cells were cultured with patient serum (50%) and analyzed for the induction of six IFN-regulated genes (LY6E, MX1, OAS1, ISG15, IFIT1, EIF2AK2) and three housekeeping genes (GAPDH, PPIB, B2M) using the Quantigene Plex 2.0 assay as described by the manufacturer (Panomics, Inc.). Increased type I IFN activity was defined as a 2-fold increase in type I IFN-regulated genes as compared to healthy controls.
7
Type I IFN Activity Measurement
8
Measuring Type I IFN Activity in SLE Serum
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Type I IFN activity was measured as previously described [36 (link), 37 (link)]. Briefly, WISH cells (CCL-25; American Type Culture Collection, Manassas, VA, USA) were cultured for 6 h with patient serum after which lysis mixture (Panomics Inc., Fremont, CA, USA) was added. Cell lysates were analyzed on a Luminex 100 (Luminex Corporation, Austin, TX, USA) for mRNA expression of three house-keeping genes (GAPDH, PPIB, B2M) and six type I IFN-regulated genes (LY6E, MX1, OAS1, ISG15, IFIT1 and EIF2AK2) using the Quantigene Plex 2.0 assay as described by the manufacturer (Panomics Inc.). The type I IFN activity was calculated by the mean fold change of the type I IFN-regulated genes in WISH cells exposed to SLE serum as compared to unstimulated WISH cells.
9
Evaluating Antioxidant Efficacy in Human Islets
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Human islets from three different donors were cultured and incubated with dh404 at 250 and 1000 nM in Dimethyl Sulphoxide Hybri-MAX (DMSO; 0.1% w/v, Sigma, St. Louis, MO) for 12 hours. Cells were then washed with PBS, pelleted, and stored at -80°C until analysis. Samples were analyzed by the Quantigene Plex 2.0 assay from Panomics (an Affymetrix company) per manufacturer's protocol. Data were normalized to the housekeeping gene POLR2A and presented as fold vehicle. Data were analyzed by one-way ANOVA followed by Duncan’s post-hoc test.
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