Gene Expression Analysis by qRT-PCR

RNA extraction was performed using RNeasy mini kit (Qiagen #74104), with DNase I treatment (Qiagen #79254). RNA concentration was measured using Nanodrop 2000c (Thermo Scientific). The cDNA synthesis was performed using 500 ng total RNA using the Takara Prime Script RT reagent kit (#RR037A). Real-time PCR was performed using the SYBR green assay at the Genomics Platform, National Center of Competence in Research Frontiers in Genetics (Geneva, Switzerland), on a 7900HT SDS system from ABI. The efficiency of each primer was assessed with serial dilutions of cDNA. Primer sequences are reported in Table 1. Relative expression levels were calculated by normalization to the geometric mean of two house-keeping genes β2-microglobulin and GAPDH as described in Vandesompele et al. (2002) (link) and expressed as relative expression values or ratio (EΔΔCt).