Vii7 real time pcr instrument

To verify overexpression of miR-9 following AAV-miR-9-eGFP microinfusion in the NAc and treatment in HEK293T cells, we used qRT-PCR. A total of 50 ng RNA extracted from NAc tissue was used for miRNA-specific reverse transcription using the miScript II RT kit (Qiagen). qRT-PCR was performed in a 384-well plate using miScript SYBR Green PCR kits (Qiagen) and miScript primer assays (Qiagen) for miR-9-5p, miR-9-3p, and RNU6B using an Applied Biosystems vii7 Real-Time PCR Instrument. For the HEK293T cells, miR-9-5p and miR-9-3p and RNU6B were measured using a TaqMan microRNA reverse transcription kit (Applied Biosystems; 20 ng RNA was used) and primer specific TaqMan Assays (Applied Biosystems). For the mRNA measurements, a total of 250 ng RNA extracted from the NAc and 100 ng RNA extracted from HEK293T cells were used for cDNA synthesis using an iScript cDNA synthesis kit (Biorad) and qRT-PCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) using primer sets designed for each mRNA (see Supplementary material). Primers for REST and OPRM1 were purchased from Qiagen. Expression data were analyzed according to the 2^−ΔΔCt method (Schmittgen and Livak, 2008 (link)).