Genechip scanner 3000 7g platform

Pooled EV RNA derived from CSF samples from two patients falling within the same age category were processed by the Clemson University Genomics Institute (N = 3 microarrays for each condition, n = 1–2 patients for each microarray). Samples were subjected to analysis on an Agilent Technologies Bioanalyzer. 500 ng of RNA was subjected to Poly A tail labeling and ligation with the FlashTag Biotin Labeling kit (Affymetrix, Santa Clara, CA) and subjected to ELOSA QC analysis. Samples were hybridized to Affymetrix Genechip miRNA 3.0 arrays (Affymetrix, Santa Clara, CA) for 16 hours at 45°C, washed, stained with an Affymetrix fluidics station 450s, and scanned using a 16-bit Genechip scanner 3000 7G platform (Solid State 532 nm Diode-pumped Frequency Doubled neodymium-doped yttrium aluminum garnet green laser). Samples were analyzed using the Affymetrix GeneChip Command Console software and TM4 microarray software suite using hierarchical clustering analysis. Alternatively, sequence analysis of miRNA was performed using Clustal Omega [24] (link). Data is deposited as a MIAME compliant study to the NCBI Gene Expression Omnibus as GEO accession number GSE57713.

Total RNA amplification and labeling was performed using the Ovation Pico WTA system V2 (NuGEN, San Carlos, CA) and Encore Biotin Module Kit (NuGEN), respectively. 5 µg of the labeled RNA from the respective samples was hybridized to GeneChip Human Transcriptome Pico Assay 2.0 microarrays (Affymetrix, Santa Clara, CA) and generated signals were finally analyzed through Affymetrix GeneChip Scanner 3000 7 G platform. Microarray signal intensities were normalized through log2-transformed Robust Multi Array (RMA) algorithm on Expression Console 1.4.1.46 (Affymetrix). Two samples (one tonsillar cancer CD141⁺ mDCs and one benign tonsil CD1c⁺ mDCs) were removed from the data set due to low amount or diminished integrity of RNA.