Mm00447557 m1

The RNAs were then used as templates for reverse transcription polymerase chain reaction (RT-PCR) using the Superscript III reverse transcriptase kit (Invitrogen, USA) following the manufacturer’s instructions. Specific TaqMan probes for tumor necrosis factor-α (TNF-α) (Mm00447557_m1) and Interleukin-6 (IL-6) (Mm00500992_m1) (Applied Biosystems, USA) were used and real-time polymerase chain reaction (rt-PCR) analysis was performed with a 7500 Fast Real-Time PCR system using TaqMan Universal PCR mix at the following thermal cycling protocol: 95° C for 10 min followed by 40 cycles of 95° C for 15 s and 60° C for 1 min. The Actin beta was used as the internal control. Data were analyzed using 7500 Fast System SDS Software 1.3.1. (Applied Biosystems).

Based on the microarray analysis, reverse transcription reactions were carried out with the SuperScript III Reverse Transcriptase (RT) kit (Invitrogen, Carlsbad, CA, USA). In accordance with the manufacturer's instructions, 1 μg of total RNA and 1 μl of oligo(dT)_12–18 primers were denatured at 65°C for 5 min and subsequently chilled at 4°C. After the addition of SuperScript III RT (200 U), the reaction mix was incubated at 42°C for 60 min, followed by another 10 min at 70°C. All primer sets and TaqMan probes for experimental genes were from Applied Biosystems (Foster City, CA, USA): mouse tyrosine hydroxylase (TH) (Mm00447557_m1), mouse pyruvate carboxylase (PC) (Mm00500992_m1), mouse brain-derived neurotrophic factor (BDNF) (Mm04230607_s1), and mouse GAPDH (Mm99999915_g1). For the quantification of mRNA, TaqMan Real Time-PCR amplification reactions were carried out using an AB 7500 Fast Real-Time PCR system (Applied Biosystems). Amplifications were performed in a final volume of 20 μl, using 10 μl of TaqMan Universal PCR Master Mix UNG (Applied Biosystems), 1 μl of the corresponding primer/probe mix, and 9 μl of template cDNA (final concentration 100 ng/20 μl). Cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, and 40 cycles at 95°C for 15 s followed by 60°C for 1 min.