Total RNA, including small non-coding RNAs, was extracted using the mirVana RNA isolation kit (Ambion, Inc, Austin, TX, USA) according to manufacturer’s protocol. RNA was quantified using a ND-1000 spectrophotometer (Nanodrop Technology®, Cambridge, UK) and RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, USA). The 260/280 ratio were between 1.7 and 2.1 and the RINs (RNA Integrity Numbers) greater than 6 for all samples.
Label
Biotin
Label protocol
Total RNA (300ng) was biotin labeled using the FlashTag™ HSR RNA labeling kit for Affymetrix miRNA arrays (Genisphere LLC., Hatfield, PA, USA) according to manufacturer’s instructions
Hybridization protocol
Labeled RNA was hybridized to Affymetrix GeneChip miRNA microarrays according to manufacturer’s instructions. Wash and stain protocol FS450_0003.
Scan protocol
GeneChips were scanned using the GC 30007G scanner.
Description
CD123+ plasmacytoid dendritic cells (pDCs), CD11c+ myeloid dendritic cells (mDCs) and CD14+ monocytes were isolated using the EasySep® Human pDCs Enrichment Kit, a custom EasySep® Human mDCs Enrichment Kit (containing antibodies directed against CD3, CD19, CD14, CD16, CD19, CD25, CD34, CD56, CD66b, GlyA, CD85g, CD45RA, CD123 and dextran) and the Human CD14 positive Selection Kit (all from Stemcell Biotechnologies) respectively according to manufacturer’s instructions.
Data processing
Data was normalized in R/Bioconductor using the robust multi-array average (RMA) expression measure.
