miRNA Expression Analysis in Sickle Cell Disease

RNA was reverse transcribed using TaqMan MicroRNA Assay (Applied Biosystems, Waltham, MA, USA) and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). RT-qPCR was performed using the Roche LightCycler 480 (Roche Applied Science) and the Bio-Rad CFX96 Real-Time PCR System using TaqMan MicroRNA Assay and TaqMan Universal Master Mix II, no UNG (Applied Biosystems) [21 (link)]. We focused on miR-451a and let-7i-5p due to the role of their expression role in SCD and malaria [3 (link),9 (link)]. The RT-qPCR primers for miR-451a, let-7i-5p, and U6 (used as an internal control) were designed using Invitrogen MicroRNA Analysis and the TaqMan Assay search tool. U6 snRNA has been widely used in other studies as an internal control for exosomal miRNA [22 (link),23 (link)]. The ΔΔCT method was used to evaluate the relative fold change in miRNA expression in Excel (Microsoft Office 365) and normalized to U6 expression [24 (link)]. ΔΔCt was created using RNU6 as an endogenous control and HbAA- as the control group in Excel using the Livak method [24 (link)]. ΔΔCt values were log-transformed to normalize the data. Any ΔΔCt over three standard deviations from the mean were flagged and removed as outliers.