Mirneasy micro

RNA was isolated from classical, intermediate or non-classical monocyte subpopulations or CD14+ monocytes using total RNA isolation kits Qiagen miRNeasy Mini or miRNeasy Micro (Qiagen). RNA quality (A260/A280 ratio) and concentration was determined on a NanoVue Spectrophotometer (VWR) and reverse-transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR was performed on a Viia7 real time PCR instrument (ThermoFisher) using the 384-well block insert. We used both SYBR green and Taqman assays (list of primers provided in Supplementary Table 2) and the respective Master mixes. After 40 amplification cycles a melting curve analysis was performed for SYBR green primers. Expression levels were calculated using the ΔΔCt method normalised to the housekeeping gene RPL28. Primer sequences for SYBR green PCR analysis were used from PrimerBank and if not available designed via NCBI Primer-BLAST. All primers for SYBR green analysis were synthesized by TIB MOLBIOL and upon arrival dissolved in nuclease-free water and stored in stock solutions of 100μM. For all SYBR primers efficiencies were calculated using a standard curve and only efficiencies between 90% and 110% were considered appropriate; all the primers were used at a final concentration of 0.2μM. Assay numbers for Taqman probes are denoted in the Supplementary Table 2.