Probe quantitect pcr mix

Total RNA was extracted with TriZOL-RNEasy kits (Invitrogen), quantified, and stored at −80 °C until analysis. The total RNA (1 µg) was reverse transcribed and real time PCR was performed for each sample in triplicate as previously described (Goyal et al. 2014 ). Taqman assays were obtained from Life Technologies for GR mRNA isoforms 1B (Hs01005211_m1), 1C (Hs01010775_m1), 1D (Hs03666144_m1) and the housekeeping gene 18S (Hs99999901_s1), and these were run according to the manufacturer’s recommendations using a Probe Quantitect PCR mix (Qiagen). Designed primers for SYBR green PCR analysis of the remaining GR mRNA isoforms and the housekeeping gene 18S, together with PCR conditions and accession numbers, are shown in Supplementary Table 2, see section on supplementary data given at the end of this article. SYBR green PCR was performed using Quantitect SYBR green PCR kit (Qiagen) using a 2.0 Lightcycler amplifier (Roche). PCR products were purified, sequenced, and used to obtain standard curves for each mRNA isoform. Extrapolation of unknowns from the standard curve was performed using Prism 3 (GraphPad Software, San Diego, CA, USA), predicting unknowns from the standard curve C_t values. Data is presented as fg mRNA/ng 18S. Alternatively, the percentages of each GR mRNA isoform were estimated for each subject as mRNA isoform/sum of all mRNA isoforms × 100.