diagnosis: rheumatoid arthritis patient status: non responder patient cell type: CD8+ T lymphocytes cell type: effector cells
Treatment protocol
Samples were stored at -80 °C until the assays were performed.
Growth protocol
Perypheral blood mononuclear cells (PBMCs) were isolated from healthy donors and patients, and CD8+ T cells subsets of interest (specific for apoptotic epitopes) were sorted with FACS AriaIII.
Extracted molecule
total RNA
Extraction protocol
The RNeasy Plus Micro Kit (Qiagen, Hilden, Germany) was used to extract RNA from 130 sorted cells from all samples.
Label
biotin
Label protocol
The nCounter® Single Cell Human Immunology v2 Kit contains fluorescent barcoded probes that are complementary to both 571immune-response and inflammation–associated genes, and 13 housekeeping genes (used as internal controls).
Hybridization protocol
In accordance with the experimental protocol, 4 μL of total RNA was incubated with 1 μL of SuperScript VILO Master Mix (Invitrogen, Life Technologies, Carlsbad, CA) at 25°C for 10 minutes, then at 42°C for 60 minutes, and finally at 85°C for 5 minutes to perform RNA reverse transcription (RT). Then, cDNA was denaturated at 94°C for 10 minutes and, after the addition of 1 μL of pooled MTE
Scan protocol
To quantify mRNA targets, each cartridge was scanned in 550 field-of-view by means of the automatic nCounter Digital Analyzer primers and 5 μL of TaqMan Pre Amp Master Mix (Applied Biosystems, Life Technology), amplified by 18 PCR cycles at 94°C for 15 seconds and 60°C for 4 minutes. After denaturation (94°C for 2 minutes, then on ice), the amplification product was hybridized for 18 hours at 65°C in the presence of both reporter and capture probes. The nCounter Prep Station was then used to purify and immobilize the resulting hybrid onto the internal surface of a cartridge.
Description
sample 9
Data processing
Raw count data were preprocessed with the NanoStringNorm R package, a set of tools for normalizing, running diagnostics, and visualizing NanoString nCounter data. Specifically, geometric mean-based scaling normalization was performed to account for technical assay variation, which involved choosing the geo.mean setting in the CodeCount R option. The background subtracted from each sample was calculated by setting mean.2sd in the background option. The method used to normalize for RNA content (i.e., pipetting fluctuations) entailed selecting the “housekeeping.geo.mean” setting in the SampleContent R option via all internal annotated housekeeping genes. All data were grouped in four classes based on cell sample subtype: naïve healthy donors (N-HDs), naïve patients (N-Ps), effector cells in healthy donors (Eff-HDs), and effector cells in patients (Eff-Ps). We did not consider genes that are not expressed in any sample, thereby excluding them from further analysis. This resulted in an analysis set of 401 unique genes and 26 samples.
