Total rna isolation nucleo spin rna 2 kit

Total RNA was isolated from the striatum and hippocampus of BDNF +/+ and BDNF -/-mice at P7 using the Total RNA Isolation Nucleospin RNA II Kit (Macherey-Nagel, Düren, Germany). Purified RNA (500 ng) was reverse transcribed using the PrimeScript™ RT reagent Kit (Perfect Real Time; Takara Biotechnology (DALIAN) CO., LTD, Japan). The cDNA synthesis was performed at 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 min in a final volume of 20 μl. The cDNA was then analyzed by quantitative real-time-PCR using a TaqMan® Gene Expression Assay (Applied Biosystems, Foster City, CA) for PTPN5 (Mm00479063_m1). Q-PCR was performed in a final volume of 12.5 μl using the Premix Ex Taq™ (Probe qPCR) (Takara Bio INC). Reactions included 40 cycles of a two-step PCR: 95 °C for 5 s and 60 °C for 20 s, after initial denaturation at 95 °C for 30 s. All Q-PCR assays were performed in duplicate. To provide negative controls, and exclude contamination by genomic DNA, the reverse transcriptase was omitted in the cDNA synthesis step. The data were analyzed and quantified using the Comparative Quantitation Analysis program of the MxProTM Q-PCR analysis software version 3.0 (Stratagene) with the 18S gene expression (PrimeTime Std qPCR Assay; Integrated DNA Technologies; Coralville, IA) as internal loading control. Results were expressed relative to wild-type values.