Quantitative RT-PCR

Brain tissue from the frontal cortex (FC), hippocampus (Hp), striatum (Str), and amygdala (Amy) was collected by gross dissection from two separate cohorts of male C57BL/6J mice exposed to the UCMS procedure. In brief, a Trizol (Life Sciences cat. No 15596-026)/chloroform-based extraction method was used to isolate total RNA. Samples were homogenized using a Kontes Pellet Pestle. Quantification of the isolated RNA was performed using the Nanodrop spectrophotometer and ND-1000 software at the optical densities of 260 and 280 nm. Samples with poor RNA quality/degradation were excluded at this stage. Reverse transcriptase amplification of cDNA from total RNA was performed using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems), conducted in a PTC-100 thermal cycler (MJ, Research). Taqman Gene Expression Assays (Applied Biosystems) were used in the Applied Biosystems StepOne Plus RT-PCR system to quantify the following target genes during amplification: prodynorphin (Pdyn) Mm00457573_m1, Oprk1 (mouse) Mm01230885_m1, Oprm1 Mm01188089_m1 and the endogenous control Rn18S (mouse) Mm04277571_s1. For each sample the average of the triplicate cycle numbers at threshold crossing (C_T) value for the endogenous control was subtracted from the average C_T values for the target gene, generating ΔC_T values. As the PCR efficiencies for both the endogenous control gene and target gene were equal (~1), the changes in expression of the target gene were expressed as 2-ΔΔC_T for each sample calculated. All data were normalized to the non-stress/saline control group and is expressed as fold change for statistical analysis and presentation.