Superscript first strand synthesis system for reverse transcription pcr rt pcr

In order to assess the release of in ammatory mediators further, qRT-PCR was introduced to determine the mRNA expression levels of myeloperoxidase (MPO), Interlaukin (IL)-1β, and IL-6. Brie y, total RNA of frozen kidneys was extracted from tissues with TRIzol Reagent (Invit-rogen, Carlsbad, CA, USA) and RNase-Free DNase I (Qiagen, Duesseldorf, Germany). The SuperScript First-Strand Synthesis System for reverse transcription PCR (RT-PCR) (Invitrogen, Carlsbad, CA, USA) was applied to synthesise cDNAs, and RNA and cDNA concentrations and purities were measured via BIO-RAD spectrophotometry (SmartSpecTM Plus, BIO-RAD, CA, USA). The primers (Table 1) were designed using Primer Premier 6.0 software and were synthesised by Shanghai Biological Engineering Co., Ltd. (Shanghai, China). PCR ampli cations were conducted using the Power SYBR® Master Mix (Invitrogen, Carlsbad, CA, USA) in an iQTM 5 Real-time PCR system (BIO-RAD, CA, USA). Expression levels were assessed relative to that of bactin, as an internal standard. Relative quanti cation of the target gene expression levels was conducted using the 2 -∆∆Ct method.