Quantitative RT-PCR Analysis of CHRNA4 Expression

The qRT-PCR analyses were performed using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) with iTaq Universal SYBR Green Supermix (Bio-Rad). All mouse and human brain and liver RNA was purchased from ZYAGEN. 500 ng total RNA was used in a 20ul reverse transcription reaction. The cDNA obtained was diluted to a total volume of 100ul and stored at −20 °C. The primers for human CHRNA4 and mouse Chrna4 (listed in Additional file 8: Table S3) were synthesized by Integrated DNA Technologies. The qRT-PCR was performed in a 20ul reaction mixture consisting of 2ul diluted cDNA, 0.2uM of each primer, and 10ul iTaq Universal SYBR Green Supermix. All amplifications were carried out in a Bio-Rad CFX96 Real-Time PCR Detection (Bio-Rad) with denaturation at 95 °C for 30s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30s. A melting curve analysis was performed for each run to confirm the specificity of amplification and lack of primer dimers. The qRT-PCR experiments were always run in triplicate. The relative mRNA expression levels of target genes were quantified using the 2-ΔΔCT methods as reported [45 (link)].