microRNA changes during Dex-induced pDC apoptosis

To identify microRNA changed during Dex-induced pDC apoptosis and CpG hampered Dex-induced pDC apoptosis Fresh peripheral blood mononuclear cells (PBMCs) were obtained from 20 healthy individuals. PDCs were isolated by magnetic-activated cell sorting using the Human Diamond Plasmacytoid Dendritic Cell Isolation Kit (Miltenyi Biotec). The purity of the isolated PDCs was > 95%, as determined by flow cytometry (FCM). Purified human pDCs were divided into three parts: 1) Medium alone, 2)Dex stimulation,3)Dex and CpG stimulation. Total RNA was harvested using TRIzol (Invitrogen) and an miRNeasy mini kit (QIAGEN) according to the manufacturers’s instructions. After confirming an adequate RNA concentration using the NanoDrop 1000, the samples were labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the miRCURY™ LNA Array (v.16.0). Following the washing steps the slides were scanned using the Axon GenePix 4000B microarray scanner. Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=50 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. The Dex induction of each miRNA was calculated by the value in pDC Dex stimulation-sample divides the value in ' pDC no stimulation-sample'. The CpG induction of each miRNA during Dex stimulation was calculated by the value in pDC Dex+CpG stimulation-sample divides the value in ' pDC Dex stimulation-sample'.