Sybr green 1 master kit

Spinal cord samples from separate cohorts of mice were dissected and stored as described above. For real-time quantitative PCR, total RNA was isolated from spinal cord tissue using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The purity and concentration were determined spectrophotometrically. Reverse transcription was accomplished using a First Strand complementary DNA Synthesis Kit (Invitrogen). Real-time PCR was performed in an ABI prism 7900HT system (Applied Biosystems). All PCR experiments were performed using the SYBR Green I master kit (Applied Biosystems). All the primers were purchased from SABiosciences (SABiosciences). Quantification was accomplished according to the standard curve method. In order to achieve the same PCR efficiency for each analyte, 1:10 serial dilutions of cDNA were used to construct standard curves for Mpdz and GAPDH. The R² values for the standard curves for each analyte approached 1.0, suggesting the same amplification efficiency in the PCR reactions under these conditions. Melting curves were performed to document single product formation and agarose electrophoresis confirmed product size. As negative controls, RNA samples that were not reverse transcribed were run. Data were normalized to GAPDH mRNA expression.

Validation by ChIP-qPCR was performed by targeting two housekeeping genes, actin5C and TER94, which typically show significant enrichment of H3K27ac in Drosophila (http://www.modencode.org/). For this assay we also targeted an intergenic region located 2 Kb upstream of these two genes as a negative control. Reactions were performed using SYBR Green I Master kit (Applied Biosystems) in an Applied Biosystems Lightcycler. The PCR parameters are: 1 cycle of 10 min at 95°C, 40 cycles of 10 s at 95°C, 10 s at 60°C, and 20 s at 72°C. PCR primer sequences are listed in Table S1.

Ten nanograms of total RNA was reverse-transcribed with miRNA-specific primers for microRNA using TaqMan microRNA Reverse Transcription kit (Applied Biosystems) according to the manufacture’s protocol. The miRNA was reversed transcribed by incubation at 16 °C for 30 min, 42 °C for 40 min, 85 °C for 5 min, and held at 4 °C to stop the reaction. Quantitative PCR assays (SYBR Green I master kit, Applied Biosystems) were performed using primers for miR-365 (GSP: GGGTAATGCCCCTAAAAAT, R: CAGTGCGTGTCGTGGAG) with the following conditions: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 10 s, and 60 °C for 60 s on a 7300HT thermocycler (Applied Biosystems). U6 was used for normalization (Forward: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, Reverse: 5′ CGCTTCACGAATTTGCGTGTCAT-3′).
For detecting β-arrestin 2 mRNA expression, PCR amplifications were performed at 94 °C for 2 min, followed by 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 90 s using primer pairs for β-arrestin 2 min, Forward: 5′-AGCACCGCGCAGTACAAGT-3′, Reverse: 5′-CACGCTTCTCTCGGTTGTCA-3′). GAPDH (Forward: 5′-AAGGTGAAGGTCGGAGTCAAC-3′, Reverse: 5′-CATGAGTCCTTCCACGATACC-3′) was used as a loading control. All samples were run in duplicate. The relative expression level of mRNA was calculated by the 2^−ΔΔCt method64 (link)65 (link).