June 2, 2022 – Gene Analysis was performed by the Clariom S Assay tool. This machine serves as a next generation transcriptome-wide gene-level expression profiling tool, which allows for the fastest, simplest, and most scalable path to generating actionable results. Based on industry-leading microarray technology, the novel rat Clariom S Assay provides extensive coverage of all known well-annotated genes, compatibility with clinical sample types, scalable formats, and flexible data analysis software. The GeneChip™ Rat Genome 230 2.0 Assay was used to evaluate gene expression levels and in cell lines that were treated with heroin and psilocybin and with psilocybin in combination with heroin.

The immortalized rat hypothalamus cells (RCHT-1) were derived from the hypothalamus of a 4-month-old Fisher Rat. The hippocampal cells (H19-7) derived from the rats’ hippocampi were treated daily with heroin and psilocybin, alone or in combination. After 48 hours, cell lines were collected for RNA extraction and gene array analysis. For each of the comparisons indicated, an electronic file was created that includes differentially expressed transcripts that were identified in both H19-7 and RCTH-1 cells with extended annotations. A second file was created that made comparisons evidencing only the transcript differences where the Ct (or cell type) value was highly different as a final analysis. For each of these data sets, only the genes whose expression was reverted by psilocybin and in the treatments consisting of heroin plus psilocybin were selected. Moreover, only the genes whose expression was psilocybin dose dependent were considered. In summary, the potential candidate genes whose expression in heroin treatment was reverted by psilocybin and evidenced in the electronic file reports were selected.