VTA Gene Expression Profiling in Mice

Mice were decapitated and their brains were removed immediately. Coronal brain slices (1 mm) were cut  using a mouse brain matrix/slicer (Braintree Sci., Inc., MA). The VTA area from each slice was then punched out using a well-polished tissue punch needle. VTA punches were homogenized and total RNA was extracted with a total RNA rapid extraction kit. HiScript II QRT SuperMix (Vazyme) was used to generate cDNA according to the following procedure: 300 ng of total RNA and 4 × gDNA wiper mix were incubated at 42 °C for 2 min to remove genome contamination, then 5 × HiScript II QRT SuperMix was added to the reaction mixture and incubated at 25 °C for 10 min, 50 °C for 30 min, and 85 °C for 5 min. The resulting cDNA was used for real-time PCR detection using a StepOnePlus real-time PCR system (Applied Biosystems, Waltham, MA, USA). The primer sequences used to amplify each product were as follows: AdipoR1 exon 2: forward-5′-CCCGTATCCACCAGACACCGG-3′; reverse-5′-GGCAATGGGGCTCCTTCTGG-3′ [31 (link)], mouse β-actin: forward-5′-GATCATTGCTCCTCCTGAGC-3′, reverse-5′-ACTCCTGCTTGCTGATCCAC-3′ [33 (link)]. The ΔΔCT method was used to obtain relative fold-change of target gene expression normalized by the housekeeping gene β-actin compared with control samples.