host strain: C57BL/6J serum source strain: K/BxN mouse model: serum transfer arthritis (STA) model time point: day 0 (Non Arthritic) tissue: synovium
Extracted molecule
total RNA
Extraction protocol
Synovial samples were isolated from the region of the tibiotalar joint and processed for RNA. Extraction of total RNA was performed according to the manufacturer's instructions for mirVaNa miRNA isolation kit (Ambion, AM1560)
Label
Rox
Label protocol
500ng of RNA samples were converted to cDNA using TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Foster City, CA). The RNA was converted to cDNA using the following conditions: 16°C for 20 min, 42°C for 1 min, and 50°C for 1 second total 40 cycles. Then, 85°C for 5 min and 4°C hold until further processing or storage.
Hybridization protocol
Prior to PCR, cDNA samples were pre-amplified using Megaplex Primer Pools, Rodent Pools Set v3.0 and TaqMan PreAmp Master Mix (Life Technologies, Foster City, CA). PreAmp Master Mix and 0.2x Primers were added to the cDNA samples and pre-amplified as follows: 95°C for 10 min once, 95°C for 15 sec, 60°C for 4 min, (final two steps repeated for 14 cycles). Quantitative Real-Time PCR reactions (qRT-PCR) were performed using the high-throughput BioMark Real-Time PCR system (Fluidigm, South San Francisco, CA). Pre-amplified cDNA samples were diluted with 0.1 mM EDTA in TE Buffer (1:5) and mixed with TaqMan Gene Expression Master Mix (Life Technologies, Foster City, CA), and Sample Loading Reagent (Fluidigm), then pipetted into sample inlets of Dynamic Array 96.96 chips (Fluidigm). Assay Loading Reagent (Fluidigm) and TaqMan miRNA Assays (Fluidigm, South San Francisco, CA) were mixed and pipetted into assay inlets of Dynamic Array 96.96 chips. This platform allows PCR reactions for 96 samples and 96 primers simultaneously. The IFC Controller HX (Fluidigm) was used to distribute primers and samples into chip reaction wells for qRT-PCR by microfluidic delivery. Real-Time PCR reactions are performed in BioMark System and PCR protocols was 95°C for 10 min denaturation 95°C for 15 seconds and 60°C for 1 min for 30 cycles.
Scan protocol
n/a
Description
Non-Art pool1
Data processing
The raw data was normalized by global normalization method (Mestdagh et al., 2009). Prior to normalization, those miRNA assays with poor or no amplification across all samples were removed. The mean Ct values across all detectable miRNAs for each sample were subtracted from individual miRNA assay Ct values to generate normalized ΔCt values. The quality of the data was assessed using Principal component Analysis and Pearson correlation to determine replicate efficacy (Spotfire DecisionSite).