Superscript 3 first strand synthesis kit

Itih3 gene expression was studied in tissue samples dissected out on an ice-cold platform. The lobe of the right liver (inferior part) as well as the hypothalamus, which is among the brain regions with the highest Itih3 gene expression [16 ], were used for analyses. Tissue samples were homogenized in lysis buffer and total RNA was isolated (mirVana RNA Isolation Kit with TURBO DNAse; Ambion, Austin, TX, USA). The quality/concentration of RNA was checked using NanoDrop (Wilmington, DE, USA). Total RNA (0.4–1 μg) was reverse-transcribed into cDNA with random hexamer primers using the Superscript III First Strand Synthesis Kit (Life Technologies, Grand Island, NY, USA) in a thermal cycler. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green Master Mix in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with primers from Integrated DNA Technologies (Coralville, IA, USA) for Itih3 (Forward 5′−CCCGGCGCATTTATGAAGAT-3′; Reverse 5′-ATGGCGTTCTCGGGGTATTC-3′) and the house keeping gene succinate dehydrogenase complex, subunit A, flavoprotein (SDHA; Forward 5′−CCTGTCCTATGTGGACGTTG-3′, Reverse 5′-GTTTTGTCGATCACGGGTCT-3′). Relative Itih3 gene expression was calculated using crossing point (Cp) values and experimentally determined PCR efficiency for Itih3 and SDHA.

RNA extracted (described above) from PD, PD-D, and control tissues was processed with the SuperScript III First-Strand Synthesis kit (Life Technologies, Carlsbad, CA) for cDNA synthesis, per the manufacturer's protocol. Quantitative real-time (qRT) PCR was completed using the Roche LightCycler 480 with SYBR Green detection.