Prism 7300 real time pcr detection system

Frozen kidneys were homogenized and total RNA was then extracted with Trizol (Sigma-Aldrich, USA) and treated with DNAse (Promega, Belgium). Total RNA concentration was measured by NanoDrop (NanoDrop 1000, Thermo Scientific, USA). Transcript-specific primers were generated on the basis of mouse sequences from GenBank. NCBI Primer Blast was used to ensure specificity of primers for each target. All pairs of primers were analysed for dissociation curves and melting temperatures. Real-time quantitative PCR was performed in order to quantify mRNA level of eNOS, iNOS, nNOS, IL-1β, IL-6, TNFα, Col I, Col III, CTGF, Periostin, TGFβ, Smad3 and 18S as a housekeeping gene (see Table 1). Briefly, 2 μg of total RNA were used for reverse transcription using MLV reverse transcriptase (Promega, Belgium) during 1 h at 70°C. Quantitative PCR amplification was performed using the SYBR Green Master Mix (Roche, Belgium) and the Prism 7300 Real-Time PCR Detection System (Applied Biosystems, CA, USA). Mean fold changes were calculated by averaging the duplicate measurement for each gene. Relative gene expressions were calculated using the 2^−ΔΔCT method.

The total RNA was extracted from the kidneys using a kit (Fermentas, Glen Burnie, MD, USA) according to the manufacturer's protocol. Real-time PCR amplification was performed using the SYBR Green master mix (ABI, USA) and the Prism 7300 real-time PCR detection system (Applied Biosystems). Oligonucleotide sequences were provided by Invitrogen and the primer pairs were presented in Table 1 [10 (link)]. The expression of mRNA levels was normalized by subtracting the corresponding GAPDH as a control and calculated by using the comparative cycle threshold method.