Ambion retroscript first strand synthesis kit

Total RNA was isolated from the plantaris muscles using the Trizol-based method, as previously described [25 (link)]. cDNA was subsequently generated from 1 μg of RNA using the Ambion RETROscript first strand synthesis kit (Life Technologies, Grand Island, NY) and used as a template for quantitative RT-PCR using a 7300 real-time PCR system (Applied Biosystems, Foster City, CA). mRNA expression was assessed using TaqMan probe-based chemistry and the following primers from Applied Biosystems: atrogin-1, GenBank NM_133521; MuRF1, GenBank NM_080903; and 18S, GeneBank X03205.1). Quantification of expression was performed using the relative standard curve method.

We isolated total RNA from human diaphragm tissue with Trizol reagent. We then used Ambion RETROscript First Strand Synthesis Kit (Life Technologies, Carlsbad, CA, USA) to generate cDNA from 1 μg of RNA. The cDNA was then used as template for qRT-PCR (7300 real-time PCR system, Applied Biosystems, Austin, TX). We used TaqMan® PCR assay primers from Life Technologies targeting the following genes and NCBI Reference Sequence numbers: Nox2 (CYBB, NM_000397.3), p47^phox (NCF1 NM_000265.5), Rac1 (RAC1, NM_006908.4), p22^phox (CYBA, NM_000101.3), p67^phox (NCF2, NM_000433.3), and p40^phox (NCF4, NM_000631.4). Gene expression quantification was performed using the relative standard-curve method, and all data were normalized to the gene expression of 18S (GeneBank NM_X03205.1) and reported relative to the control group.