Quantifying Rat Brain mRNA Levels

Quantification of mRNA was conducted using reverse transcription PCR and primers specifically designed for rat mRNAs Lsd1, Lsd1 + 8a, and Bdnf exon IV (Table 1). RNA was isolated from amygdala tissue as described previously (Kyzar et al., 2017 (link), 2019 (link)) and then reverse transcribed in duplicate using mixed random primers and MuLV reverse transcriptase (Life Technologies). Quantitative real-time PCR was performed using either the Mx3000P qPCR system (Agilent Technologies) and SYBR Green master mix (Fermentas) or a CFX Connect qPCR system with iQ SYBR SuperMix (Bio-Rad). Data were analyzed using the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)). Hprt1 was used as a reference gene (Kyzar et al., 2017 (link), 2019 (link)). Data are represented as fold change compared to control AIS groups.