Real time quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

qRT-PCR assays were developed to determine the level of messenger RNA (mRNA) expression of different genes using SYBR Green (Applied Biosystems, Warrington, United Kingdom). Forward and reverse primers (Sigma-Aldrich, St. Louis, MO) were designed from different exons in order to eliminate DNA contamination (see supplemental Table 1 on the Blood Web site). Actin was used as endogenous control. Human GATA1 and human pri-miR-486 expression were examined using TaqMan gene expression assay and RPLPO was used as endogenous control (Applied Biosystems). Samples were tested in duplicate on the Applied Biosystems 7900HT Fast Real-Time PCR System.