**2.10. Quantitative reverse transcription-PCR (qRT-PCR) analysis of MicroRNAs**

To screen for potential microRNAs contributing to the ketamine-induced neurotoxicity, we used human miFinder miRNA PCR arrays (Qiagen) which screen 84 microRNAs. For the arrays, a master mix (25 μL/well) containing the template cDNA (4.5 ng/well), RNase-free water, universal primer and miScript SYBR Green (Qiagen) was prepared according to the manufacturer's instructions. The primers for each microRNAs to be analyzed come lyophilized in the wells of the array plates. To confirm the array results, validation assays were performed in which the three cDNA samples used for the arrays were each run in triplicate on the same PCR run. The PCR was run using a BioRad iCycler for 15 min at 95°C followed by 40 cycles of: denaturation (15 s at 94°C), annealing (30 s at 55°C) and extension (30 s at 70°C). Melt curve and reverse transcriptase controls were run to ensure sample purity and primer specificity, respectively. The epoch nanodrop spectrophotometer was also used to assess RNA quality using the A260:A280 ratio. To be considered pure, the A260:A280 for an RNA sample must fall between 1.8 and 2.2. Samples with an A260:A280 ratio outside of this accepted range were not used for the studies. MicroRNAs that displayed a 1.3 fold change in expression between the ketamine and control treated cells were considered to be of interest.
