Quantitative Real-Time PCR for Gene Expression Analysis

One microgram of total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Life Technologies, Applied Biosystems, Foster City, CA, USA). Real-time PCR reactions were performed in duplicates using the TaqMan Universal PCR Master Mix II with no UNG (Life Technologies, Applied Biosystems) on 50 ng of the resulting cDNA, with an ABI PRISM 7900HT thermocycler under the following conditions: 10 min at 95°C, 50 cycles of 15 s at 95°C and 1 min at 60°C. Primers/TaqMan probe assays purchased from Applied Biosystems were used to determine the level of expression of the mouse and human candidate genes (Supplementary Table S2). A search into the microarray data for probes showing stable expression in the stressed, non-stressed and Flx-treated animals but also blood, DG and ACC, revealed Rab5a as a moderately expressed reference gene. For human expression, we used CRYL1 as a reference gene for highly/moderately expressed genes and SV2A for weakly expressed genes (Belzeaux et al., 2012 (link); Supplementary Table S2). Raw Ct values were obtained with manual baseline settings on the RQ Manager software (Applied Biosystems), and then the relative expression level of each mRNA was quantified by using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)).