SH-SY5Y cells, primary human neurons, or primary rat neurons were exposed to various concentrations of morphine (0.1, 1.0, 5.0 μM) based on experimental designs. Cells were harvested at various time points, and total RNA was extracted with an RNA extraction kit (New England Biolabs) according to the manufacturer’s instructions. Total RNA samples were also extracted from postmortem brain tissues from PWH and various brain regions of F344 control and HIV-1-Tg rats. RT-PCR reactions were performed as described previously (Donadoni et al. 2019 (link)). Briefly, RNA samples were first treated with DNase I followed by phenol/chloroform extraction and ethanol precipitation. cDNAs were synthesized by using M-MuLV reverse transcriptase and RNA templates were removed from reactions by RNase H digestion. A total of 100 ng cDNA was used as template for PCR reactions. Alternatively, spliced isoforms of OPRM1 gene were amplified by PCR using primers listed on Table 1. β-Actin and GAPDH mRNA were also amplified from the same set of RNA samples by RT-PCR as internal controls. Amplified gene products were resolved on 1– 1.5% DNA-agarose gels and visualized by ethidium bromide staining.