Luxscan 10k a

Plasma samples from 15 patients and 12 controls were analyzed using an antigen microarray (GeneCopoeia, USA). The 120 IgG- and IgM-specific self-antigens that were analyzed are listed in Supplemental Table S7. Briefly, serum samples were first heat-inactivated and pretreated with DNAse I. Then, the samples were diluted 1:100 in PBS and applied to the arrays on the slide for hybridization with the antigens. The binding of IgG and IgM antibodies to antigens in the array was detected with Cy3-conjugated anti-human IgG (1/1000) and Cy5-conjugated anti-human IgM (1/1000) secondary antibodies. The slides were scanned with a LuxScan 10 K-A (CapitalBio Corporation, China) with laser wavelengths of 532 (for IgG) and 635 (for IgM) nm. Images in the array were converted to raw data with LuxScan_v3.0 (CapitalBio Corporation, China). The net fluorescent intensity (NFI) of each antigen was generated by subtracting the local background and negative control (PBS) signals. The signal/noise ratio (SNR = [foreground median - background median]/SD [background]) was calculated for each antigen. The NFI was normalized by a robust linear model using positive controls with different dilutions.

The microarray ISAC 112 (ThermoFisher, Uppsala, Sweden) is a solid phase fluoro immunoassay that detects IgE antibodies against the proteins fixed on ISAC surface. One slide contains 4 microarray and one kit 5 slides, 20 microarrays in total. The technique was performed following the manufacturer's instructions. Each microarray is incubated with a serum in order to label sIgE to each protein and subsequently it is incubated with a human anti IgE detection antibody conjugated with fluorescence.
Finally the fluorescence intensity of each microarray is measured by the scanner (LuxScan 10K/A, CapitalBio, Beijing, China) using following parameters: laser power (LP = 60) and photo-multiplier tube (PMT = 600).
The analysis of the digitalized images is performed with the software Phadia Microarray Image Analyzer (ThermoFisher). This software allows transforming the images fluorescence intensity in numerical data according to the calibration curve built with the calibrator CTR02 sample included in each assay, as previously described (figure 1). An acceptable calibration curve needs to show slope parameters (Y) between 5.5 and 6.8, and R²>0.85 according to the information provided by ThermoFisher.
The sIgE values are expressed semiquantitatively as ISU. Results equal or greater than 0.30 ISU are considered positive, according to the indications of the manufacturer.

Total RNA was extracted from cultured cells by using the TRIzol reagent (Invitrogen, CA) and purified by using NucleoSpin® RNA clean-up Kit (Macherey-Nagel, Germany). Then, RNA integrity was evaluated under the 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). Subsequently, hybridization was performed overnight on a 22K human genome array chip, V1.0 (CapitalBio Corp., Beijing, China), based on the manufacturer’s instructions. After that, the hybridization images were obtained using the LuxScan 10KA dual-channel laser scanner and digitized with LuxScan 3.0 image analysis software (CapitalBio Corp.). Finally, microarray data were collected and filtered by subtracting the background. The experiment was performed in biologic triplicates with three technical replicates. The genes dysregulated at least two-fold in comparison with the controls, with a false-discovery rate <5%, were collected for further analysis.

To identify differences in miRNA profile between the 5-8F/Si-control and the 5-8F/Si-c-Myc cell lines, the miRNA microarray analysis was performed using a miRNA microarray obtained from from Capital Bio Corporation (Beijing, China) and composed of 434 human (containing 122 predicted miRNA sequences from a published reference), 196 rat and 261 mouse miRNAs that were registered in the Sanger miRBase miRNA database (http://www.mirbase.org/; miRBase Release 8.2). The miRNA microarray used was a single-channel fluorescence chip with all oligonucleotide probes being labelled with Cy3 fluorescent dyes (green colour). The miRNAs were enriched from total RNA-extracted cells (5-8F/Si-c-Myc and 5-8F/Si-control) using a mirVana miRNA Isolation Kit (Ambion, Foster City, CA) and labelled using a mirVana Array Labelling Kit. Labelled miRNAs were then hybridized to miRNA microarrays that had 509 probes in triplicate to determine differential expression between the cell lines. This procedure was repeated twice. Fluorescence scanning was performed using a double-channel laser scanner (LuxScan 10K/A; CapitalBio). Figure signals were transformed to digital signals using image analysis software (LuxScan3.0; CapitalBio). Raw data were normalized and analyzed using the Significance Analysis of Microarrays software (SAM, version 2.1; Stanford University, CA).