Tac 4.0

Total RNA from adrenal glands of 2-month-old animals was extracted using the Total RNA Mini Kit (FroggaBio) according to the manufacturer's protocol. RNA (n = 3 per group) was submitted to the McGill University and Génome Québec Innovation Centre (Montreal, Quebec, Canada) for RNA quality control and microarray analysis using the Affymetrix Clariom S Mouse assay. Results were analyzed using Transcriptome Analysis Console software (TAC 4.0) (Thermo Fisher Scientific). Thresholds used for P value and fold change were .05 and ±2, respectively. Further analyses were performed using the Metascape gene annotation and analysis resource [18 (link)]. A protein–protein interaction profile was additionally performed using the string database [19 (link)].

Differences in miRNA expression levels were analyzed using the TAC4.0 (Thermo Fisher Scientific Inc.) and JMP pro 13.0 software package for Windows (SAS Institute Inc.).
Expression levels of miRNAs were analyzed using Mann-Whitney U test. We determined the cutoff expression levels using receiver operating characteristic (ROC) analysis. We calculated disease-free survival (without recurrence including metachronous metastasis) from the date of surgery to the development of recurrence (including metachronous metastasis) or the last follow-up, and overall survival from the date of surgery to death or the last follow-up using Kaplan-Meier analysis. A log rank test was conducted after Kaplan-Meier analysis to determine significance. Potential factors associated with survival were identified by univariate and multivariate analyses using Cox proportional hazards regression models conducted using JMP 13.0 software. P<0.01 and P<0.05 were considered significant in array analysis and non-array analysis, respectively.