Genome-wide microRNA analysis of reticulocytes isolated from patients with sickle cell disease

MiRNAs are non-protein-coding small RNA molecules negatively regulating gene through inhibition of mRNA. The characteristics of microRNA expression patterns obtained from sickle cell diseases are not clearly elucidated. In this study, we recruited 12 children and adults suffering from sickle cells diseases with high HbF fetal hemoglobin (HbF) group (6 subjects) and lower HbF (6 subjects). The peripheral blood mononuclear cell (PBMN) were processed using a MACS column with magnetic anti-CD71 antibody to isolate reticulocytes according to the manufacturer’s instructions (Miltenyi Biotec, San Diego, CA). After data normalization, 327 miRNA were identified by PCA (principal component analysis) as differentially expressed in low HbF group compared to high HbF groups. Twelve samples were isolated from six sickle cell disease (SCD) patients with high HbF and six with low HbF levels were analyzed. Informed consent was obtained from the subjects and the study was approved by the Institutional Review Boards. Five milliliters of whole-blood were collected from the SCD peripheral blood in EDTA tubes. Following Histopaque separation under centrifuge condition 500g for 30 min, the CD71+ cells located in a layer below peripheral blood mononuclear cell were isolated using a MACS column with magnetic anti-CD71 antibody to isolate reticulocytes according to the manufacturer’s instructions (Miltenyi Biotec, San Diego, CA). Total RNA was extracted from the CD71+ cell by Trizol solution according to the manufacturer’s instructions. After dissolving in 30 μl of RNase free water, the RNA concentration (A260 nm) and purity (A260/280 and A260/230 ratios) were assessed with a ND-1000 spectrophotometer (Thermo Scientific, Wilmington DE) and quality measurement was assayed by Experion equipment (BIO-RAD, Hercules, CA). The RNA was stored at −80°C until further analysis. Subsequently, RNA was isolated from CD71+ cell and used for microRNA Array (Exiqon) containing 1921 human probes for 568 microRNAs mined by two colour data after normalization.