The Expression Patterns of Reference Genes in the Brains of Male Rats Subjected to CUMS-induced Depressive-like Behavior Strongly Depends on both the Age and the Specific Brain Region.

The objective of this investigation was to identify the most suitable candidates for RT-qPCR analysis. The study focused on the medial prefrontal cortex (mPFC), ventral (VH) and dorsal (DH) hippocampi of rats (1.5, 10, and 20 months old) subjected to a seven-week chronic unpredictable mild stress (CUMS) paradigm to induce depressive-like behavior (DLB). The mRNA expression stability of eight widely employed housekeeping genes was evaluated using the comprehensive RefFinder algorithm. Stability of the candidate genes was found to vary considerably depending on both brain region and animal age. In the mPFC, no single gene was stable across all ages, necessitating age-stratified analysis; the most stable pairs were Tbp/Ywhaz in young, Helz/Gapdh in adult, and Eef1a1/Ppia in elderly rats. In the VH, optimal pairs were Pgk1/Helz (young), Ppia/Eef1a1 (adult), and Ppia/Pgk1 (elderly). For the DH, Pgk1/Gapdh was most stable for young and adult rats, while Gapdh/Hmbs was optimal for the elderly group. Our findings demonstrate the necessity of validating reference genes under specific experimental conditions. We provide validated, region- and age-specific gene sets for use in rats with CUMS-induced DLB, establishing a methodological framework for reliable normalization in depression research.